Methionine sulfone and S-substituted cysteine sulfone derivatives as enzyme inhibitors

ABSTRACT

This invention relates to compounds which inhibit thrombin or factor Xa. The compounds contain an aldehyde functionality and a methionine sulfone or S-substituted cysteine sulfone residue. The compounds and their pharmaceutical compositions are useful for preventing thrombosis in mammals which are suspected of having a condition characterized by abnormal thrombosis.

RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/234,811, filed Apr.28, 1994 which is a continuation-in-part of U.S. Ser. No. 08/229,298,filed Apr. 18, 1994 which is hereby incorporated by reference herein,including the drawings attached thereto.

TECHNICAL FIELD

In one aspect, the present invention relates compounds which are potentinhibitors of thrombin or factor Xa. In another aspect, the presentinvention relates to novel peptide aldehyde analogs, theirpharmaceutically acceptable salts, and pharmaceutically acceptablecompositions thereof which are useful as potent and specific inhibitorsof blood coagulation in vitro and in vivo in mammals. In yet anotheraspect, the invention relates to methods of using these inhibitors astherapeutic agents for disease states in mammals characterized byabnormal thrombosis.

BACKGROUND

Normal hemostasis is the result of a complex balance between theprocesses of clot formation (blood coagulation) and clot dissolution(fibrinolysis). The complex interactions between blood cells, specificplasma proteins and the vascular surface, maintain the fluidity of bloodunless injury and blood loss occur.

Blood coagulation is the culmination of a series of amplified reactionsin which several specific zymogens of serine proteases in plasma areactivated by limited proteolysis. Nemerson, Y. and Nossel, H. L., Ann.Rev. Med., 33:479 (1982). This series of reactions results in theformation of an insoluble fibrin matrix which is required for thestabilization of the primary hemostatic plug. The interaction andpropagation of the activation reactions occurs through the extrinsic andintrinsic pathways of coagulation.

These pathways are highly inter-dependent and converge in the formationof the serine protease, Factor Xa. Factor Xa catalyzes the penultimatestep in the blood coagulation cascade which is the formation of theserine protease thrombin. This step occurs following the assembly of theprothrombinase complex which is composed of factor Xa, the non-enzymaticco-factor Va and the substrate prothrombin assembled on the surface ofadhered, activated platelets or systemically circulating membranousmicroparticles.

Proteolytic activation of zymogen factor X to its catalytically activeform, factor Xa, can occur by either the intrinsic or extrinsiccoagulation pathways.

The intrinsic pathway is referred to as "intrinsic" because everythingneeded for clotting is in the blood. Saito, H., "Normal HemostaticMechanisms", Disorders of Hemostasis, pp. 27-29, Grune & Stratton, Inc.(O. D. Ratnoff, M. D. and C. D. Forbes, M. D. edit. 1984). This pathwayis comprised of the zymogen serine proteases, factors IX and XI, and thenon-enzymatic co-factor, factor VIII. The initiation of the intrinsicpathway results in the activation of factor XI to XIa. Factor XIacatalyzes the activation of factor IX to factor IXa which in combinationwith the activated form of factor VIII on an appropriate phospholipidsurface, results in the formation of the tenase complex. This complexalso catalyzes the formation of the serine protease, factor Xa, from itszymogen, factor X which subsequently results in clot formation.

The extrinsic pathway is referred to as "extrinsic" because the tissuefactor which binds to and facilitates the activation of factor VII comesfrom outside the blood. Saito, Id. The major components of this pathwayare the zymogen serine protease, factor VII, and the membrane boundprotein, tissue factor. The latter serves as the requisite non-enzymaticco-factor for this enzyme. The initiation of this pathway is thought tobe an autocatalytic event resulting from the activation of zymogenfactor VII by trace levels of activated factor VII (factor VIIa), bothof which are bound to newly exposed tissue factor on membrane surfacesat sites of vascular damage. The factor VIIa/tissue factor complexdirectly catalyzes the formation of the serine protease, factor Xa, fromits zymogen, factor X. Exposure of blood to injured tissue initiatesblood clotting by the extrinsic pathway.

The formation of thrombin is catalyzed by factor Xa following theassembly of the catalytic prothrombinase complex as reviewed by Mann, K.G. et. al., "Surface-Dependent Reactions of the Vitamin K-DependentEnzyme Complexes", Blood, 76:1-16 (1990). This complex is composed offactor Xa, the non-enzymatic co-factor Va and the substrate prothrombinall assembled on an appropriate phospholipid surface. The requirement ofa macromolecular complex for efficient catalysis results in theprotection of factor Xa from natural anticoagulant mechanisms such asheparin-antithrombin III mediated inhibition. Teite, J. M. andRosenberg, R. D., "Protection of Factor Xa from neutralization by theheparin-antithrombin complex", J. Clin. Invest., 76:1383-1391 (1983). Inaddition, sequestration of factor Xa in the prothrombinase complex alsorenders it resistant to inhibition by exogenous heparin therapy whichalso requires antithrombin III to elicit its anticoagulant effect.

Thrombin is the primary mediator of thrombus formation. Thrombin actsdirectly to cause formation of insoluble fibrin from circulatingfibrinogen. In addition, thrombin activates the zymogen factor XIII tothe active transglutaminase factor XIIIa which acts to covalentlystabilize the growing thrombus by crosslinking the fibrin strands.Lorand, L. and Konishi, K., Arch. Biochem. Biophys., 105:58 (1964).Beyond its direct role in the formation and stabilization of fibrin richclots, the enzyme has been reported to have profound bioregulatoryeffects on a number of cellular components within the vasculature andblood. Shuman, M. A., Ann. New York Acad. Sci., 405:349 (1986).

It is believed that thrombin is the most potent agonist of plateletactivation, and it has been demonstrated to be the primarypathophysiologic-mediator of platelet-dependent arterial thrombusformation. Edit, J. F. et al., J. Clin. Invest., 84:18 (1989).Thrombin-mediated platelet activation leads to ligand-inducedinter-platelet aggregation principally due to the bivalent interactionsbetween adhesive ligands such as fibrinogen and fibronectin withplatelet integrin receptors such as glycoprotein IIb/IIIa which assumetheir active conformation following thrombin activation. Berndt, M. C.and Phillips, D. R., Platelets in Biology and Pathology, pp 43-74,Elsevier/North Holland Biomedical Press (Gordon, J. L. edit. 1981).Thrombin-activated platelets can also support further thrombinproduction through the assembly of new prothrombinase and tenase (factorIXa, factor VIIIa and factor X) catalytic complexes on the membranesurface of intact activated platelets and platelet-derivedmicroparticles, following thrombin-mediated activation of thenon-enzymatic cofactors V and VIII, respectively. Tans, G. et al.,Blood, 77:2641 (1991). This positive feedback process results in thelocal generation of large concentrations of thrombin within the vicinityof the thrombus which supports further thrombus growth and extension.Mann, K. G. et al., Blood, 76:1 (1990).

In contrast to its prothrombotic effects, thrombin has been shown toinfluence other aspects of hemostasis. These include its effect as animportant physiological anticoagulant. The anticoagulant effect ofthrombin is expressed following binding of thrombin to the endothelialcell membrane glycoprotein, thrombomodulin. This is thought to result inan alteration of the substrate specificity of thrombin thereby allowingit to recognize and proteolytically activate circulating protein C togive activated protein C (aPC). Musci, G. et al., Biochemistry, 27:769(1988). aPC is a serine protease which selectively inactivates thenon-enzymatic co-factors Va and VIIIa resulting in a down-regulation ofthrombin formation by the prothrombinase and tenase catalytic complexes,respectively. Esmon, C. T., Science, 235:1348 (1987). The activation ofprotein C by thrombin in the absence of thrombomodulin is poor.

Thrombin has also been shown to be a potent direct mitogen for a numberof cell types, including cells of mesenchymal origin such as vascularsmooth muscle cells. Chen, L. B. and Buchanan, J. M., Proc. Natl. Acad.Sci. USA, 72:131 (1975). The direct interaction of thrombin withvascular smooth muscle also results in vasoconstriction. Walz, D. A. etal., Proc. Soc. Expl. Biol. Med., 180:518 (1985). Thrombin acts as adirect secretagogue inducing the release of a number of bioactivesubstances from vascular endothelial cells including tissue plasminogenactivator. Levin, E. G. et al., Thromb. Haemost., 56:115 (1986). Inaddition to these direct effects on vascular cells, the enzyme canindirectly elaborate potent mitogenic activity on vascular smooth musclecells by the release of several potent growth factors (e.g.platelet-derived growth factor and epidermal growth factor) fromplatelet a-granules following thrombin-induced activation. Ross, R., N.Engl. J. Med., 314:408 (1986).

Many significant disease states are related to abnormal hemostasis. Withrespect to the coronary arterial vasculature, abnormal thrombusformation due to the rupture of an established atherosclerotic plaque isthe major cause of acute myocardial infarction and unstable angina.Moreover, treatment of an occlusive coronary thrombus by eitherthrombolytic therapy or percutaneous transluminal coronary angioplasty(PTCA) is often accompanied by an acute thrombotic reclusure of theaffected vessel which requires immediate resolution. With respect to thevenous vasculature, a high percentage of patients undergoing majorsurgery in the lower extremities or the abdominal area suffer fromthrombus formation in the venous vasculature which can result in reducedblood flow to the affected extremity and a predisposition to pulmonaryembolism. Disseminated intravascular coagulopathy commonly occurs withinboth vascular systems during septic shock, certain viral infections andcancer and is characterized by the rapid consumption of coagulationfactors and systemic coagulation which results in the formation oflife-threatening thrombi occurring throughout the vasculature leading towidespread organ failure.

Pathogenic thrombosis in the arterial vasculature is a major clinicalconcern in today's medicine. It is the leading cause of acute myocardialinfarction which is one of the leading causes of death in the westernworld. Recurrent arterial thrombosis also remains one of the leadingcauses of failure following enzymatic or mechanical recanalization ofoccluded coronary vessels using thrombolytic agents or percutaneoustransluminal coronary angioplasty (PTCA), respectively. Ross, A. M.,Thrombosis in Cardiovascular Disorder, p. 327, W. B. Saunders Co.(Fuster, V. and Verstraete, M. edit. 1991); Califf, R. M. and Willerson,J. T., Id. at p 389. In contrast to thrombotic events in the venousvasculature, arterial thrombosis is the result of a complex interactionbetween fibrin formation resulting from the blood coagulation cascadeand cellular components, particularly platelets, which make up a largepercentage of arterial thrombi. Heparin, the most widely used clinicalanticoagulant administered i.v., has not been shown to be universallyeffective in the treatment or prevention of acute arterial thrombosis orrethrombosis. Prins, M. H. and Hirsh, J., J. Am. Coll. Cardiol., 67:3A(1991).

Besides the unpredictable, recurrent thrombotic reocclusion whichcommonly occurs following PTCA, a profound restenosis of the recanalizedvessel occurs in 30 to 40% of patients 1 to 6 months following thisprocedure. Califf, R. M. et al., J. Am. Coll. Cardiol., 17:2B (1991).These patients require further treatment with either a repeat PTCA orcoronary artery bypass surgery to relieve the newly formed stenosis.Restenosis of a mechanically damaged vessel is not a thrombotic processbut instead is the result of a hyperproliferative response in thesurrounding smooth muscle cells which over time results in a decreasedluminal diameter of the affected vessel due to increased muscle mass.Id. As for arterial thrombosis, there is currently no effectivepharmacologic treatment for the prevention of vascular restenosisfollowing mechanical recanalization.

The need for safe and effective therapeutic anticoagulants has in oneaspect focused on the role of the serine protease thrombin in bloodcoagulation.

Most preferred natural substrates for thrombin are reported to containan uncharged amino acid in the P3 recognition subsite. For example, thethrombin cleavage site on the Aα chain of fibrinogen, which is theprimary physiological substrate for thrombin, is reported to contain aglycine residue in this position while the cleavage site on the Bβ chaincontains a serine, as shown below:

    ______________________________________                                        P4 P3 P2 P1 P1'                                                               Gly--Gly--Val--Arg/Gly                                                                            Fibrinogen Aα Chain                                 Phe--Ser--Ala--Arg/Gly                                                                            Fibrinogen Bβ Chain                                  ______________________________________                                    

Peptidyl derivatives having an uncharged residue in the P3 position aresaid to bind to the active site of thrombin and thereby inhibit theconversion of fibrinogen to fibrin and cellular activation have beenreported. These derivatives have either an aldehyde, chloromethyl ketoneor boronic acid functionality associated with the P1 amino acid. Forexample, substrate-like peptidyl derivatives such asD-phenylalanyl-prolyl-argininal (D-Phe-Pro-Arg-al),D-phenylalanyl-prolyl-arginine-chloromethyl ketone (P-PACK) andacetyl-D-phenylalanyl-prolyl-boroarginine (Ac-(D-Phe)-Pro-boroArg) havebeen reported to inhibit thrombin by directly binding to the active siteof the enzyme. Bajusz, S., Symposia Biologica Hungarica, 25: 277 (1984),Bajusz, S. et al, J. Med. Chem., 33:1729 (1990) and Bajusz, S. et al.,Int. J. Peptide Protein Res. 12:217 (1970); Kettner, C. and Shaw, E.,Methods Enzymol., 80:826 (1987), Kettner, C. et al., EP 293,881(published Dec. 7, 1988), Kettner, C., et al., J. Biol. Chem., 265:18209(1990). These molecules have been reported to be potent anticoagulantsin the prevention of platelet-rich arterial thrombosis. Kelly, A. B. etal., Thromb. Haemostas., 65:736 at abstract 257 (1991). Other peptidylaldehydes have been proposed or reported as inhibitors of thrombin. Bey,P. et al., EP 363,284 (published Apr. 11, 1990) and Balasubramanian, N.et al., EP 526,877 (published Feb. 10, 1993).

Peptidyl compounds which are said to be active site inhibitors ofthrombin but which differ in structure from those containing a unchargedamino acid in the P3 recognition subsite have been reported.

The compound, Argatroban (also called 2R,4R-4-methyl-1-[N-2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-argininal]-2-piperdinecarboxylicacid), is also reported to bind directly to the active site of thrombinand has been thought to be the most potent and selective compound in theclass of non-peptidyl inhibitors of this enzyme. Okamoto, S. et al.,Biochem. Biophys. Res. Commun., 101:440 (1981). Argatroban has beenreported to be a potent antithrombotic agent in several experimentalmodels of acute arterial thrombosis. Jang, I. K. et al., in bothCirculation, 81: 219 (1990) and Circ. Res., 67:1552 (1990).

Peptidyl compounds which are said to be inhibitors of thrombin and whosemode of action is thought to be by binding to both the active site andanother site on the enzyme have been reported. Hirudin and certainpeptidyl derivatives of hirudin have been reported to inhibit bothconversion of fibrinogen to fibrin and platelet activation by binding toeither both the active site and exo site, or the exo site only, ofthrombin. Markwardt, F., Thromb. Haemostas., 66:141 (1991). Hirudin isreported to be a 65 amino acid polypeptide originally isolated fromleech salivary gland extracts. It is said to be one of the most potentinhibitors of thrombin known. Marki, W. E. and Wallis, R. B., Thromb.Haemostas., 64:344 (1990). It has been reported to inhibit thrombin bybinding to both its anion-binding exo-site and to its catalytic activesite which are distinct and physically distant from each other. Rydel,T. J. et al., Science, 249:277 (1990). Hirudin has been reported to be apotent antithrombotic agent in vivo. Markwardt, F. et al., Pharmazie,43:202 (1988); Kelly, A. B. et al., Blood, 77:1 (1991). In addition toits antithrombotic effects, hirudin has been reported to alsoeffectively inhibit smooth muscle proliferation and the associatedrestenosis following mechanical damage to a atherosclerotic rabbitfemoral artery. Sarembock, I. J. et al., Circulation, 84:232 (1991).

Hirugen has been reported to be a peptide derived from the anioniccarboxy-terminus of hirudin. It is reported to bind only to the anionbinding exo-site of thrombin and thereby inhibit the formation of fibrinbut not the catalytic turnover of small synthetic substrates which haveaccess to the unblocked active site of the enzyme. Maraganore, J. M. etal., J. Biol. Chem., 264:8692 (1989); Naski, M. C. et al., J. Biol.Chem., 265:13484 (1990). The region of hirudin represented by hirugenhas been reported, as according to by x-ray crystallographic analysis,to bind directly to the exo site of thrombin. Skrzypczak-Jankun, E. etal., Thromb. Haemostas., 65:830 at abstract 507 (1991). Moreover, thebinding of hirugen has also been reported to enhance the catalyticturnover of certain small synthetic substrates by thrombin, indicatingthat a conformational change in the enzyme active site may accompanyoccupancy of the exo-site. Liu, L. W. et al., J. Biol. Chem, 266:16977(1991). Hirugen also is reported to block thrombin-mediated plateletaggregation. Jakubowski, J. A. and Maraganore, J. M., Blood, 72:399(1990).

A group of synthetic chimeric molecules comprised of a hirugen-likesequence linked by a glycine-spacer region to the peptide,D-phenylalanyl-prolyl-arginine, which is based on a preferred substraterecognition site for thrombin, has been termed to be hirulog. Maraganoreet al., U.S. Pat. No. 5,196,404 (Mar. 23, 1993). The hirugen-likesequence is said to be linked to this peptide through the C-terminal endof the peptide. Maraganone, J. M. et al., Biochemistry, 29:7095 (1990).The hirulogs have been reported to be an effective antithrombotic agentsin preventing both fibrin-rich and platelet-rich thrombosis. Maraganone,J. M. et al., Thromb. Haemostas., 65:651 at abstract 17 (1991).

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to compounds which areuseful in vitro and in vivo as antithrombic agents.

According to a preferred aspect, the peptide aldehydes of the presentinvention include those depicted in formula (I) below: ##STR1## wherein(a) X is selected from the group consisting of --C(O)--, --S(O₂)--,--O--S(O₂)--, --NH--S(O₂)-- and --N(R')--S(O₂)--, wherein R' is alkyl of1 to about 4 carbon atoms, aryl of about 6 to about 14 carbon atoms, oraralkyl of about 6 to about 15 carbon atoms;

(b) R₁ is selected from the group consisting of:

(1) alkyl of about 3 to about 10 carbon atoms,

(2) alkyl of 1 to about 3 carbon atoms substituted with cyclic alkyl ofabout 5 to about 8 carbon atoms,

(3) alkenyl of about 3 to about 6 carbon atoms,

(4) alkenyl of about 3 to about 6 carbon atoms which is substituted withcyclic alkyl of about 5 to about 8 carbon atoms,

(5) aryl of about 6 to about 14 carbon atoms,

(6) aryl of about 6 to about 14 carbon atoms which is substituted withY₁,

(7) aryl of about 6 to about 14 carbon atoms which is substituted withY₁ and Y₂,

(8) aralkyl of about 6 to about 15 carbon atoms,

(9) aralkyl of about 6 to about 15 carbon atoms which is substituted inthe aryl ring with Y₁,

(10) aralkyl of about 6 to about 15 carbon atoms which is substituted inthe aryl ring with Y₁ and Y₂,

(11) aralkenyl of about 8 to about 15 carbon atoms,

(12) aralkenyl of about 8 to about 15 carbon atoms which is substitutedin the aryl ring with Y₁,

(13) aralkenyl of about 8 to about 15 carbon atoms which is substitutedin the aryl ring with Y₁ and Y₂,

(14) perfluoroalkyl of 1 to about 12 carbon atoms,

(15) perfluoroaryl of about 6 to about 14 carbon atoms,

(16) trimethylsilylalkyl of about 4 to about 8 carbon atoms, ##STR2##wherein W is oxygen, methylene, --C(O)--, --CH(OH)--, --CH (OA₁)--,--CH(C(O)--OH)--, --CH(C(O)--OR')--, --CH(C(O)--NHR')--,--CH(C(O)--NR'R")--, ##STR3## --NH-- or --N (R')--, and X is not--O--S(O₂)--, --NH--S (O₂)-- or --N(R') --S(O₂)--,

(22) ##STR4## where X is not --O--S(O₂)--, --NH--S (O₂)-- or--N(R')--S(O₂)--,

(23) ##STR5## wherein W' is oxygen, sulfur, --S(O)--, --S(O₂)--,methylene, --C(O)--, --CH(OH)--, --CH(OA₁)--, --CH(C(O)--OH)--,--CH(C(O)--OR')--, --CH(C(O)--NHR')--, --CH(C(O)--NR'R")--, ##STR6##--NH-- or --N(R')--, and X is not --O--S(O₂)--, --NH--S(O₂)-- or--N(R')--S(O₂)--,

(24) ##STR7## where X is not --O--S(O₂)--, --NH--S(O₂)-- or--N(R')--S(O₂)--,

(25) ##STR8## where X is not --O--S(O₂)--, --NH--S (O₂)-- or--N(R')--S(O₂)--,

(26) ##STR9## where X is not --O--S(O₂)--, --NH--S(O₂)-- or--N(R')--S(O₂)--,

(27) a substituted group of the formula ##STR10## wherein the aryl ringis substituted with Y₁, and X is not --O--S(O₂)--, --NH--S(O₂)-- or--N(R')--S(O₂)--, and

(28) a substituted group of the formula ##STR11## wherein the aryl ringis substituted with Y₁ and Y₂, and X is not --O--S(O₂)--, --NH--S(O₂)--or --N(R')--S (O₂)--,

wherein Y₁ and Y₂ are independently selected from the group consistingof bromo, chloro, fluoro, --Z₁, --OH, --OZ₁, --NH₂, --NHZ₁, --NZ₁ Z₂,--NH--C(O)--Z₁, N(Z₁)--C(O)--Z₂ --NH--C(O)--OZ₁, --N(Z₁)--C(O)--OZ₂,--NH--C(O)--NH₂, --NH--C(O)--NHZ₁, --NH--C(O)--NZ₁ Z₂,--N(Z₁)--C(O)--NHZ₂, --N (Z₁)--C(O)--NZ₂ Z₃, --C(O)--OH, --C(O)--OZ₁,--C(O)--NHZ₁, --C(O)--NZ₁ Z₂, --SH, --SZ₁, --S(O)--Z₁, --S(O₂)--Z₁,--S(O₂)--OH, --S (O₂)--OZ₁, --S(O₂)--NH₂, --S(O₂)--NHZ₁, --S(O₂)--NZ₁ Z₂and ##STR12## wherein Z₁, Z₂ and Z₃ are independently selected from thegroup consisting of trifluoromethyl, pentafluoroethyl, alkyl of 1 toabout 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, andaralkyl of about 6 to about 15 carbon atoms,

R" is alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about 14carbon atoms, or aralkyl of about 6 to about 15 carbon atoms,

A₁ is aryl of about 6 to about 14 carbon atoms, aryl of about 6 to about14 carbon atoms which is substituted with Y₁, aralkyl of about 6 toabout 15 carbon atoms, or aralkyl of about 6 to about 15 carbon atomswhich is substituted with Y₁ ;

(c) R₂ is selected from the group consisting of

    --CH.sub.2 --S--CH.sub.3,

    --CH.sub.2 --S(O)--CH.sub.3,

    --CH.sub.2 --S(O.sub.2)--CH.sub.3,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NR'R",

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NR'R",

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NR'R", ##STR13##

    --S--CH.sub.3,

    --S(O)--CH.sub.3,

    --S(O.sub.2)--CH.sub.3,

    --S--(CH.sub.2).sub.m --C(O)--OH,

    --S(O)--(CH.sub.2).sub.m --C(O)--OH,

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OH,

    --S--(CH.sub.2).sub.m --C(O)--OR',

    --S(O)--(CH.sub.2).sub.m --C(O)--OR',

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OR',

    --S--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S(O)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S--(CH.sub.2).sub.m --C(O)--NHR',

    --S(O)--(CH.sub.2).sub.m --C(O)--NHR',

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NHR',

    --S--(CH.sub.2).sub.m --C(O)--NR'R",

    --S(O)--(CH.sub.2).sub.m --C(O)--NR'R",

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NR'R", ##STR14## wherein m is 1, 2, 3, 4, 5 or 6; and (d) n is 1, 2 or 3; or pharmaceutically acceptable salts thereof.

Peptidyl arginine aldehydes have been reported to exist in equilibriumstructures in aqueous solutions. Bajusz, S., et al., J. Med. Chem.,33:1729 (1990). These structures, as shown below, include the argininealdehyde, A, aldehyde hydrate, B, and two amino cyclol forms, C and D.The R group would represent the remainder of a given compound embodiedin the present invention. The peptide aldehydes of the present inventioninclude within their definition all the equilibrium forms. ##STR15##

In another aspect, the present invention is directed to pharmaceuticalcompositions comprising a therapeutically effective amount of a compoundof the present invention and a pharmaceutically acceptable carrier.

In yet another aspect, the present invention is directed to methods ofusing the compounds and pharmaceutical compositions of the presentinvention for the prevention of thrombosis in a mammal suspected ofhaving a condition characterized by abnormal thrombosis, comprisingadministering to said mammal a therapeutically effective amount of acompound the present invention or pharmaceutical composition comprisingsuch a compound.

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined to have following meanings, unlessexplicitly stated otherwise.

The term "alkyl" refers to saturated aliphatic groups includingstraight-chain, branched-chain and cyclic groups.

The term "alkoxy" refers to a group having the formula, R--O--, whereinR is an alkyl group.

The term "alkenyl" refers to unsaturated aliphatic groups which containat least one carbon-carbon double bond and includes straight-chain,branched-chain and cyclic groups.

The term "alkenyloxy" refers to a group having the formula, R--O--,wherein R is an alkenyl group.

The term "aryl" refers to aromatic groups which have at least one ringhaving a conjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups, all of which may be optionallysubstituted.

The term "aryloxy" refers to a group having the formula, R--O--, whereinR is an aryl group.

The term "aralkyl" refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,all of which may be optionally substituted.

The term "aralkoxy" refers to a group having the formula, R--O--,wherein R is an aralkyl group.

The term "aralkenyl" refers to an alkenyl group substituted with an arylgroup. Suitable aralkenyl groups include styrenyl and the like, all ofwhich may be optionally substituted.

The term "aralkenyloxy" refers to a group having the formula, R--O--,wherein R is an aralkenyl group.

The term "alkylene" refers to a divalent straight chain or branchedchain saturated aliphatic radical.

The term "alkylenecarboxy" refers to the group --alk--COOH where alk isalklene.

The term "carboxamide" refers to the group --C(═O)--NH₂.

The term "alkylenecarboxamide" refers to the group --alk--C(═O)--NH₂where alk is alkylene.

The term "alkylenehydroxy" refers to the group --alk--OH wherein alk isalkylene.

The term "amino acid" refers to both natural and unnatural amino acids.Natural amino acids include alanine (Ala), arginine (Arg), asparagine(Ash), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Set), threonine (Thr), tryptophan (Trp), tyrosine (Tyr)and valine (Val). Unnatural amino acids include, but are not limited toazetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminoisobutyricacid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylglycine, N-methylisoleucine, N-methylvaline, norvaline,norleucine, ornithine and pipecolic acid.

The term "amino acid residue" refers to --NH--CH(R)--CO--, wherein R isthe side chain group distinguishing each amino acid. For cyclic aminoacids, the residue is ##STR16## wherein x is 1, 2 or 3 representing theazetidinecarboxylic acid, proline or pipecolic acid residues,respectively.

The term "methylene" refers to --CH₂ --.

The term "perfluoroalkyl" refers to an alkyl group wherein each hydrogenis replaced by a fluoro. Suitable perfluoroalkyl groups includeperfluoromethyl (having the structure of CF₃ --) and perfluroethyl(having the structure of CF₃ --CF₂ --) and the like.

The term "perfluoroaryl" refers to an aryl group wherein each hydrogenis replaced by a fluoro. Suitable perfluoroaryl groups includeperfluorophenyl (having the formula of ##STR17## and 2-perfluoronaphthyl(having the formula of ##STR18## and the like.

In addition, the following abbreviations stand for the following:

"N-Boc-N^(g) -nitro-L-arginine" refers to the compound which has theformula: ##STR19##

"Arg-al" refers to L-argininal which has the formula: ##STR20##

"Arg-ol" refers to L-argininol which has the formula: ##STR21##

"Azt" refers to L-2-azetidinecarboxylic acid.

"Bzl" refers to benzyl.

"BzlSO₂ " refers to benzylsulfonyl.

"Boc" refers to t-butoxycarbonyl.

"BOP" refers tobenzotriazol-1-yloxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate.

"Brine" means an aqueous saturated solution of sodium chloride.

"BuSO₂ " refers to 1-butanesulfonyl.

"d-camphor--SO₂ " refers to d-10-camphorsulfonyl which has the formula:##STR22##

"1-camphor--SO₂ " refers to 1-10-camphorsulfonyl which has the formula:##STR23##

"CDI" refers to carbonyldiimidazole.

"ChxCH₂ SO₂ " refers to cyclohexylmethanesulfonyl.

"ChxNHSO₂ " refers to N-cyclohexylaminosulfonyl.

"2-CMPhSO₂ " refers to 2-carbomethoxy-1-phenylsulfonyl.

"Cys[S--CH₃ ]" refers to S-methyl-L-cysteine which has the formula:##STR24##

"Cys [S(O)--CH₃ ]" refers to S-methyl-L-cysteine sulfoxide which has theformula: ##STR25##

"Cys [S(O₂)--CH₃ ]" refers to S-methyl-L-cysteine sulfone which has theformula: ##STR26##

"Cys [S--CH₂ CO₂ H]" refers to S-(carboxymethyl)-L-cysteine which hasthe formula: ##STR27##

"Cys [S(O)--CH₂ CO₂ H]" refers to S-(carboxymethyl)-L-cysteine sulfoxidewhich has the formula: ##STR28##

"Cys [S(O₂)--CH₂ CO₂ H]" refers to S-(carboxymethyl)-L-cysteine sulfonewhich has the formula: ##STR29##

"Cys[S-CH₂ CO₂ CH₃ ] refers to S-(carbomethoxymethyl)-L-cysteine whichhas the formula: ##STR30##

"Cys [S(O)--CH₂ CO₂ CH₃ ] refers to S-(carbomethoxymethyl)-L-cysteinesulfoxide which has the formula: ##STR31##

"Cys[S(O₂)--CH₂ CO₂ CH₃ ] refers to S-(carbomethoxymethyl)-L-cysteinesulfone which has the formula: ##STR32##

"Cys [(S--CH₂ Tzl] refers to S-(tetrazol-5-ylmethyl)-L-cysteine whichhas the formula: ##STR33##

"Cys [S(O)--CH₂ Tzl] refers to S-(tetrazol-5-yl)-L-cysteine methylsulfoxide which has the formula: ##STR34##

"Cys [S(O₂)--CH₂ Tzl] refers to S-(tetrazol-5-yl)-L-cysteine methylsulfone which has the formula: ##STR35##

"DCC" refers to 1,3-dicyclohexylcarbodiimide.

"DCM" refers to dichloromethane.

"DIEA" refers to diisopropylethylamine.

"DMF" refers to N,N-dimethylformamide.

"EDC" refers to ethyl-3-(3-dimethylamino)-propylcarbodiimidehydrochloride salt.

"HCl" refers to hydrochloric acid.

"HF" refers to hydrofluoric acid.

"HOBt" refers to 1-hydroxybenzotriazole monohydrate. "MeOH" refers tomethanol. "Met[S(O)]" refers to L-methionine sulfoxide which has theformula: ##STR36##

"Met[S(O₂)]" refers to L-methionine sulfone which has the formula:##STR37##

"MgSO₄ " refers to anhydrous magnesium sulfate.

"MeOH" refers to methanol.

"1-NpSO₂ " refers to 1-naphthalenesulfonyl.

"2-NpSO₂ " refers to 2-naphthalenesulfonyl.

"NMM" refers to 4-methylmorpholine.

"Ph" refers to phenyl.

"Pip" refers to L-pipecolic acid.

"Pro" refers to L-proline.

"2-PrPen" refers to 2-propylpentanoyl.

"TBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate.

"TEA" refers to triethylamine.

"TFA" refers to trifluoroacetic acid.

"THF" refers to tetrahydrofuran.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a reaction scheme describing a process for preparing asolid-phase reagent which may be used to make the compounds of thepresent invention. In this figure, Bzl refers to benzyl; t-Bu refers tot-butyl; and Boc refers to t-butoxycarbonyl.

FIG. 2 depicts the anticoagulant effect of the compound of Example 27,BzlSO₂ -Met[S(O₂)]-Pro-Arg-al, measured in citrated rat () and human(∘) plasma using the activated partial thromboplastin time (APTT) assay.The elevation in APTT due to increasing concentration of compound ispresented relative to the control clotting time for rat (19.75 sec) andhuman (28.3 sec) plasma which is set to a value of 1.0.

DETAILED DESCRIPTION OF THE INVENTION

1. Preferred Compounds.

In one aspect, the present invention is directed to certain compoundswhich are the peptide aldehydes as depicted in formula (IA) below:##STR38## wherein (a) X is selected from the group consisting of--C(O)--, --S(O₂)--, --O--S(O₂)--, --NH--S(O₂)-- and --N(R')--S(O₂)--,wherein R' is alkyl of 1 to about 4 carbon atoms, aryl of about 6 toabout 14 carbon atoms, or aralkyl of about 6 to about 15 carbon atoms;

(b) R₁ is selected from the group consisting of:

(1) alkyl of about 3 to about 10 carbon atoms,

(2) alkyl of 1 to about 3 carbon atoms substituted with cyclic alkyl ofabout 5 to about 8 carbon atoms,

(3) alkenyl of about 3 to about 6 carbon atoms,

(4) alkenyl of about 3 to about 6 carbon atoms which is substituted withcyclic alkyl of about 5 to about 8 carbon atoms,

(5) aryl of about 6 to about 14 carbon atoms,

(6) aryl of about 6 to about 14 carbon atoms which is substituted withY₁,

(7) aryl of about 6 to about 14 carbon atoms which is substituted withY₁ and Y₂,

(8) aralkyl of about 6 to about 15 carbon atoms,

(9) aralkyl of about 6 to about 15 carbon atoms which is substituted inthe aryl ring with Y₁,

(10) aralkyl of about 6 to about 15 carbon atoms which is substituted inthe aryl ring with Y₁ and Y₂,

(11) aralkenyl of about 8 to about 15 carbon atoms,

(12) aralkenyl of about 8 to about 15 carbon atoms which is substitutedin the aryl ring with Y₁,

(13) aralkenyl of about 8 to about 15 carbon atoms which is substitutedin the aryl ring with Y₁ and Y₂,

(14) perfluoroalkyl of 1 to about 12 carbon atoms,

(15) perfluoroaryl of about 6 to about 14 carbon atoms,

(16) trimethylsilylalkyl of about 4 to about 8 carbon atoms, ##STR39##wherein W is oxygen, methylene, --C(O)--, --CH(OH)--, --CH(OA₁)--,--CH(C(O)--OH)--, --CH (C(O)--OR')--, --CH(C(O)--NHR')--,--CH(C(O)--NR'R")--, ##STR40## --NH-- or --N(R')--, and X is not--O--S(O₂)--, --NH--S(O₂)-- or --N(R")--S(O₂)--,

(22) ##STR41## where X is not --O--S(O₂)--, --NH--S(O₂)-- or --N(R')--S(O₂)--,

(23) ##STR42## wherein W' is oxygen, sulfur, --S(O)--, --S(O₂)--,methylene, --C(O)--, --CH(OH)--, --CH(OA₁)--, --CH(C(O)--OH)--,--CH(C(O)--OR')--, --CH(C(O)--NHR')--, --CH(C(O)--NR'R")--, ##STR43##--NH-- or --N(R')--, and X is not --O--S(O₂)--, --NH--S(O₂)-- or --N(R')--S(O₂)--,

(24) ##STR44## where X is not --O--S(O₂)--, --NH--S(O₂)-- or--N(R')--S(O₂)--,

(25) ##STR45## where X is not --O--S(O₂)--, --NH--S(O₂)-- or --N (R')--S(O₂)--,

(26) ##STR46## where X is not --O--S(O₂)--, --NH--S(O₂)-- or--N(R')--S(O₂)--,

(27) a substituted group of the formula ##STR47## wherein the aryl ringis substituted with Y₁, and X is not --O--S(O₂)--, --NH--S(O₂)-- or--N(R')--S(O₂)--, and

(28) a substituted group of the formula ##STR48## wherein the aryl ringis substituted with Y₁ and Y₂, and X is not --O--S(O₂)--, --NH--S(O₂)--or --N(R')--S(O₂)--,

wherein

Y₁ and Y₂ are independently selected from the group consisting of bromo,chloro, fluoro, --Z₁, --OH, --OZ₁, --NH₂, --NHZ₁, --NZ₁ Z₂,--NH--C(O)--Z₁, --N(Z₁)--C(O)--Z₂, --NH--C(O)--OZ₁, --N(Z₁)--C(O)--OZ₂,--NH--C(O)--NH₂, --NH--C(O)--NHZ₁, --NH--C(O)--NZ₁ Z₂,--N(Z₁)--C(O)--NHZ₂, --N(Z₁)--C(O)--NZ₂ Z₃, --C(O)--OH, --C(O)--OZ₁,--C(O)--NHZ₁, --C(O)--NZ₁ Z₂, --SH, --SZ₁, --S(O)--Z₁, --S(O₂)--Z₁,--S(O₂)--OH, --S(O₂)--OZ₁, --S(O₂)--NH₂, --S(O₂)--NHZ₁, --S(O₂)--NZ₁ Z₂and ##STR49## wherein Z₁, Z₂ and Z₃ are independently selected from thegroup consisting of trifluoromethyl, pentafluoroethyl, alkyl of 1 toabout 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, andaralkyl of about 6 to about 15 carbon atoms,

R" is alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about 14carbon atoms, or aralkyl of about 6 to about 15 carbon atoms,

A₁ is aryl of about 6 to about 14 carbon atoms, aryl of about 6 to about14 carbon atoms which is substituted with Y₁, aralkyl of about 6 toabout 15 carbon atoms, or aralkyl of about 6 to about 15 carbon atomswhich is substituted with Y₁ ;

(c) R₂ is selected from the group consisting of

    --CH.sub.2 --S--CH.sub.3,

    --CH.sub.2 --S(O)--CH.sub.3,

    --CH.sub.2 --S(O.sub.2)--CH.sub.3,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NR'R",

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NR'R",

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NR'R",

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NR'R", ##STR50##

    --S--CH.sub.3,

    --S(O)--CH.sub.3,

    --S(O.sub.2)--CH.sub.3,

    --S--(CH.sub.2).sub.m --C(O)--OH,

    --S(O)--(CH.sub.2).sub.m --C(O)--OH,

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OH,

    --S--(CH.sub.2).sub.m --C(O)--OR',

    --S(O)--(CH.sub.2).sub.m --C(O)--OR',

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OR',

    --S--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S(O)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S--(CH.sub.2).sub.m --C(O)--NHR',

    --S(O)--(CH.sub.2).sub.m --C(O)--NHR',

    --S(O)--(CH.sub.2).sub.m --C(O)--NHR',

    --S--(CH.sub.2).sub.m --C(O)--NR'R",

    --S(O)--(CH.sub.2).sub.m --C(O)--NR'R",

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NR'R", ##STR51## wherein m is 1, 2, 3, 4, 5 or 6; and (d) n is 1, 2 or 3; or pharmaceutically acceptable salts thereof.

Compounds of formula (IA) include those wherein P2 isL-2-azetidinecarboxylic acid, L-proline or L-pipecolic acid. Preferredcompounds include those wherein P2 is L-2-azetidinecarboxylic acid orL-proline. Especially preferred compounds include those wherein P2 isL-proline.

The compounds of formula (IA) include those wherein P3 is: (1)L-cysteine which is substituted on its sulfur atom with a methyl orstraight-chain alkylene of 1 to about 6 carbon atoms, wherein saidstraight-chain alkylene is substituted with --C(O)--OH, --C(O)--OR',--C(O)--NH₂, --C(O)--NHR', --C(O)--NR'R", or tetrazol-5-yl, wherein R'and R" are independently selected from the group consisting of alkyl of1 to about 4 carbon atoms, aryl of about 6 to about 14 carbon atoms, oraralkyl of about 6 to about 15 carbon atoms; (2) L-cysteine sulfoxidewhich is substituted on its sulfur atom with a methyl or straight-chainalkylene of 1 to about 6 carbon atoms, wherein said straight-chainalkylene is substituted with --C(O)--OH, --C(O)--OR', --C(O)--NH₂,--C(O)--NHR', --C(O)--NR'R", or tetrazol-5-yl; (3) L-cysteine sulfonewhich is substituted on its sulfur atom with a methyl or straight-chainalkylene of 1 to about 6 carbon atoms, wherein said straight-chainalkylene is substituted with --C(O)--OH, --C(O)--OR', --C(O)--NH₂,--C(O)--NHR', --C(O)--NR'R", or tetrazol-5-yl; (4) L-methionine; (5)L-methionine which is substituted on its S-methyl group with astraight-chain alkylene of 1 to about 6 carbon atoms, wherein saidstraight-chain alkylene is substituted with --C(O)--OH, --C(O)--OR',--C(O)--NH₂, --C(O)--NHR', --C(O)--NR'R", or tetrazol-5-yl; (6)L-methionine sulfoxide; (7) L-methionine sulfoxide which is substitutedon its S-methyl group with a straight-chain alkylene of 1 to about 6carbon atoms, wherein said straight-chain alkylene is substituted with--C(O)--OH, --C(O)--O--R', --C(O)--NH₂, --C(O)--NHR', --C(O)--NR'R", ortetrazol-5-yl; (8) L-methionine sulfone; or (9) L-methionine sulfonewhich is substituted on its S-methyl group with a straight-chainalkylene of 1 to about 6 carbon atoms, wherein said straight-chainalkylene is substituted with --C(O)--OH, --C(O)--O--R', --C(O)--NH₂,--C(O)--NHR', --C(O)--NR'R", or tetrazol-5-yl.

Preferred compounds include those wherein the amino acid residue at P3is: (1) L-cysteine which is substituted on its sulfur atom with astraight-chain alkylene of 1 to 3 carbon atoms, wherein saidstraight-chain alkylene is substituted with --C(O)--OH, --C(O)--O--CH₃,--C(O)--O--CH₂ --CH₃, --C(O)--O--CH₂ --CH₂ --CH₃, or tetrazol-5-yl; (2)L-cysteine sulfoxide which is substituted on its sulfur atom with amethyl or straight-chain alkylene of 1 to 3 carbon atoms, wherein saidstraight-chain alkylene is substituted with --C(O)--OH, --C(O)--O--CH₃,--C(O)--O--CH₂ --CH₃, --C(O)--O--CH₂ --CH₂ --CH₃, or tetrazol-5-yl; (3)L-cysteine sulfone which is substituted on its sulfur atom with a methylor straight-chain alkylene of 1 to 3 carbon atoms, wherein saidstraight-chain alkylene is substituted with --C(O)--OH, --C(O)--O--CH₃,--C(O)--O--CH₂ --CH₃, --C(O)--O--CH₂ --CH₂ --CH₃, or tetrazol-5-yl; (4)L-methionine; (5) L-methionine which is substituted on its S-methylgroup with a straight-chain alkylene of 1 to 3 carbon atoms, whereinsaid straight-chain alkylene is substituted with --C(O)--OH,--C(O)--O--CH₃, --C(O)--O--CH₂ --CH₃, --C(O)--O--CH₂ --CH₂ --CH₃, ortetrazol-5-yl; (6) L-methionine sulfoxide; (7) L-methionine sulfoxidewhich is substituted on its S-methyl group with a straight-chainalkylene of 1 to 3 carbon atoms, wherein said straight-chain alkylene issubstituted with --C(O)--OH, --C(O)--O--CH₃, --C(O)--O--CH₂ --CH₃,--C(O)--O--CH₂ --CH₂ --CH₃, or tetrazol-5-yl; (8) L-methionine sulfone;or (9) L-methionine sulfone which is substituted on its S-methyl groupwith a straight-chain alkylene of 1 to 3 carbon atoms, wherein saidstraight-chain alkylene is substituted with --C(O)--OH, --C(O)--O--CH₃,--C(O)--O--CH₂ --CH₃, --C(O)--O--CH₂ --CH₂ --CH₃, or tetrazol-5-yl.

Especially preferred compounds include those wherein

P3 is L-methionine sulfone, S-methyl-L-cysteine sulfone,

S-(carboxymethyl)-L-cysteine sulfone,

S(carbomethoxymethyl)-L-cysteine sulfone,

S-(carboethoxymethyl)-L-cysteine sulfone, or

S-(carbopropyloxymethyl)-L-cysteine sulfone.

Compounds of formula (IA) include those wherein P4 is R₁ --C(O)--, R₁--S(O₂)--, R₁ --O--S(O₂)--, R₁ --NH--S(O₂)-- or R₁ --N(R')--S(O₂)--.

Preferred compounds include those wherein R₁ is alkyl of about 3 toabout 10 carbon atoms; alkyl of 1 to about 3 carbon atoms substitutedwith cyclic alkyl of about 5 to about 8 carbon atoms; aryl of about 6 toabout 14 carbon atoms; aryl of about 6 to about 14 carbon atoms which issubstituted with Y₁ ; aryl of about 6 to about 14 carbon atoms which issubstituted with Y₁ and Y₂ ; aralkyl of about 6 to about 15 carbonatoms; aralkyl of about 6 to about 15 carbon atoms which is substitutedin the aryl ring with Y₁ ; aralkyl of about 6 to about 15 carbon atomswhich is substituted in the aryl ring with Y₁ and Y₂ ; ##STR52##

Suitable alkyls for R₁ include 1-propyl, 2-methyl-1-propyl,2,2-dimethyl-1-propyl, 2-propyl, 2-methyl-2-propyl, 1-butyl, 2-butyl,3-butyl, 3-methy-1-butyl, 1-pentyl, cyclopentyl, 1-hexyl,cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, 1-heptyl, 4-heptyl,octyl, nonanyl, dodecanyl, adamantyl or adamantylmethyl. Suitable cyclicalkyls include cyclopentyl, cyclopentylmethyl, cyclohexyl,cyclohexylmethyl, adamantyl and adamantylmethyl. Suitable aryl groupsfor R₁ include phenyl, 2-carboxyphenyl, 2-carbomethoxyphenyl, 2-indanyl,2-indenyl, 1-naphthyl, 2-naphthyl, 2-thienyl, 2-pyrrolyl or 2-furyl.Suitable aralkyl groups include benzyl, diphenylmethyl, biphenylmethyl,naphthylmethyl, alpha-phenylmethylphenyl and 2-phenylethylene.

Where P3 is L-methionine sulfone, preferred compounds of formula (IA)include those wherein P4 is

    R.sub.1 --S(O.sub.2)--, R.sub.1 --O--S(O.sub.2)--, R.sub.1 --NH--S(O.sub.2)-- or R.sub.1 --C(O)--.

Especially preferred compounds include those wherein P4 is

    R.sub.1 --S(O.sub.2)-- or R.sub.1 --C(O)--.

Where P4 is R₁ --S(O₂)--, preferred compounds include those wherein R₁is alkyl of about 3 to about 10 carbon atoms, alkyl of 1 to about 3carbon atoms substituted with cyclic alkyl of about 5 to about 8 carbonatoms, aryl of about 6 to about 14 carbon atoms, aryl of about 6 toabout 14 carbon atoms which is substituted with Y₁, aryl of about 6 toabout f4 carbon atoms which is substituted with Y₁ and Y₂, aralkyl ofabout 6 to about 15 carbon atoms aralkyl of about 6 to about 15 carbonatoms which is substituted in the aryl ring with Y₁, or aralkyl of about6 to about 15 carbon atoms which is substituted in the aryl ring with Y₁and Y₂. Especially preferred compounds include those wherein R₁ is arylof about 6 to about 14 carbon atoms, or aryl of about 6 to about 14carbon atoms which is substituted with Y₁. More especially preferredcompounds include those wherein R₁ is phenyl, 2-carboxyphenyl,2-carbomethoxyphenyl, 1-naphthyl or 2-naphthyl.

Where P4 is R₁ --C(O)--, preferred compounds include those wherein R₁ isalkyl of about 3 to about 10 carbon atoms, alkyl of 1 to about 3 carbonatoms substituted with cyclic alkyl of about 5 to about 8 carbon atoms,aralkyl of about 6 to about 15 carbon atoms, aralkyl of about 6 to about15 carbon atoms which is substituted in the aryl ring with Y₁, oraralkyl of about 6 to about 15 carbon atoms which is substituted in thearyl ring with Y₁ and Y₂. Especially preferred compounds include thosewherein R₁ is alkyl of about 3 to about 10 carbon atoms. More especiallypreferred compounds include those wherein R₁ is 1-butyl, 1-heptyl or4-heptyl.

Alternatively, where P3 is S-methyl-L-cysteine sulfone, preferredcompounds of formula (IA) include those wherein P4 is R₁ --S(O₂)--, R₁--NH--S(O₂)-- or R₁ --C(O)--. Especially preferred compounds includethose wherein P4 is R₁ --S(O₂)--.

Where P4 is R₁ --S(O₂)--, preferred compounds include those wherein R₁is alkyl of about 3 to about 10 carbon atoms, alkyl of 1 to about 3carbon atoms substituted with cyclic alkyl of about 5 to about 8 carbonatoms, aryl of about 6 to about 14 carbon atoms, aryl of about 6 toabout 14 carbon atoms which is substituted with Y₁, aryl of about 6 toabout 14 carbon atoms which is substituted with Y₁ and Y₂, aralkyl ofabout 6 to about 15 carbon atoms, aralkyl of about 6 to about 15 carbonatoms which is substituted in the aryl ring with Y₁, or aralkyl of about6 to about 15 carbon at6ms which is substituted in the aryl ring with Y₁and Y₂. Especially preferred compounds include those wherein R₁ is arylof about 6 to about 14, or aryl of about 6 to about 14 carbon atomswhich is substituted with Y₁. More especially preferred compound includethose wherein R₁ is phenyl, 2-carboxyphenyl, 2-carbomethoxyphenyl,1-naphthyl or 2-naphthyl.

Alternatively, where P3 is S-(carboxymethyl)-L-cysteine sulfone,S-(carbomethoxymethyl)-L-cysteine sulfone,S-(carboethoxymethyl)-L-cysteine sulfone, orS-(carbopropyloxymethyl)-L-cysteine sulfone, preferred compounds offormula (IA) include those wherein P4 is R₁ --S(O₂)--, R₁ --NH--S(O₂)--or R₁ --C(O)--. Especially preferred compounds include those wherein P3is S-(carboxymethyl)-L-cysteine sulfone orS-(carbomethoxymethyl)-L-cysteine sulfone and P4 is R₁ --S(O₂)-- or R₁--C(O)--.

Where P3 is S-(carboxymethyl)-L-cysteine sulfone orS-(carbomethoxymethyl)-L-cysteine sulfone and P4 is R₁ --S(O)--,preferred compounds include those wherein R₁ is alkyl of about 3 toabout 10 carbon atoms, alkyl of 1 to about 3 carbon atoms substitutedwith cyclic alkyl of about 5 to about 8 carbon atoms, aryl of about 6 toabout 14 carbon atoms, aryl of about 6 to about 14 carbon atoms which issubstituted with Y₁, aryl of about 6 to about 14 carbon atoms which issubstituted with Y₁ and Y₂, aralkyl of about 6 to about 15 carbon atoms,aralkyl of about 6 to about 15 carbon atoms which is substituted in thearyl ring with Y₁, or aralkyl of about 6 to about 15 carbon atoms whichis substituted in the aryl ring with Y₁ and Y₂. Especially preferredcompounds include those wherein R₁ is aryl of about 6 to about 14 carbonatoms, or aryl of about 6 to about 14 carbon atoms which is substitutedwith Y₁. More especially preferred compounds include those wherein P3 isS-(carboxymethyl)-L-cysteine sulfone and R₁ is phenyl, 1-naphthyl or2-naphthyl. More especially preferred compounds also include thosewherein P3 is S-(carbomethoxymethyl)-L-cysteine sulfone and R₁ isphenyl, 2-carboxyphenyl, 2-carbomethoxyphenyl, 1-naphthyl or 2-naphthyl.

Where P3 is S-(carboxymethyl)-L-cysteine sulfone orS-(carbomethoxymethyl)-L-cysteine sulfone and P4 is R₁ --C(O)--,preferred compounds include those wherein R₁ is alkyl of about 3 toabout 10 carbon atoms, alkyl of 1 to about 3 carbon atoms substitutedwith cyclic alkyl of about 5 to about 8 carbon atoms, aralkyl of about 6to about 15 carbon atoms, aralkyl of about 6 to about 15 carbon atomswhich is substituted in the aryl ring with Y₁, or aralkyl of about 6 toabout 15 carbon atoms which is substituted in the aryl ring with Y₁ andY₂. Especially preferred compounds include those wherein R₁ is alkyl ofabout 3 to about 10 carbon atoms. More especially preferred compoundsinclude those wherein R₁ is 1-butyl, 1-heptyl or 4-heptyl.

Certain preferred compounds of the present invention include: ##STR53##2. Preparation of Preferred Compounds.

The compounds of the present invention are synthesized by either solidor liquid phase methods. Under certain conditions, the liquid phasemethod disclosed herein is preferred.

Many of the starting materials used in either of these methods arereadily available from chemical vendors as Aldrich, Sigma, NovaBiochemicals and the like.

During the synthesis of these compounds, the functional groups of theamino acid derivatives used in these methods are protected by blockinggroups to prevent cross reaction during the coupling procedure. Examplesof suitable blocking groups and their use are described in "ThePeptides: Analysis, Synthesis, Biology", Academic Press, Vol. 3 (E.Gross & Meienhofer edit. 1981) and Vol. 9 (S. Udenfriend & J. Meienhoferedit. 1987), the disclosure of which are incorporated herein byreference.

The compounds of the present invention are synthesized by solid phaseprocedures described in the literature (see below) or by sequentialchemical attachment of amino acid derivatives using the solid phasesynthesis reagents and methods disclosed in the commonly assigned U.S.patent of Webb, T. R., U.S. Pat. No. 5,283,293 (issued Feb. 1, 1994),and the commonly assigned U.S. patent application of Webb, T. R., U.S.Ser. No. 07/807,474, filed Dec. 13, 1991, the disclosures of which areincorporated herein by reference.

FIG. 1 illustrates the synthesis of a solid phase reagent of Webb. Thisreagent is preferred for use in the solid phase method of Webb to makethe compounds of the present invention. The details of how this reagentis made is provided in Examples 1 to 7.

Example 8 provides a preferred solid phase method to make the compoundsof the present invention. In this method, the compound in its protectedform is synthesized on a solid phase resin by the sequential coupling(using BOP or TBTU in combination with HOBt) of the amino acids or aminoacid derivatives which comprise the sequence of the desired finalcompound. The compound is removed from the solid phase in the form of adeprotected semicarbazone by treatment with hydrofluoric acid/anisole.This treatment, in addition to removing the compound from the resin as asemicarbazone, deprotects by removing O-t-butyl and N-nitro protectinggroups from the amino acid or amino acid side chain groups. Thedeprotected semicarbazone is then transformed by hydrolysis to acompound of the present invention by treatment with ethylacetoacetate ina pH 1 mixture of acetic acid, tetrahydrofuran and water. Examples 11,18, 21, 24, 27, 40, 43, 46, 49, 56, 59, 66 and 69 are provided tofurther demonstrate the use of this solid phase synthesis method.

The peptide aldehydes of the present invention may also be synthesizedby solution phase methods. The preferred method is provided in Examples28 through 33. In this method, N-Boc-N^(g) -nitro-L-arginine is firstconverted to N-Boc-N^(g) -nitro-L-argininol by treatment withborane-tetrahydrofuran complex. Next, N-Boc-N^(g) -nitro-L-argininol isconverted to N^(g) -nitro-L-argininol by treatment with hydrochloricacid. Next, N^(g) -nitro-L-argininol having a free N-terminal aminogroup is coupled a pre-synthesized dipeptide having free C-terminalcarboxyl group and a blocked N-terminal amino group to givedipeptide-N^(g) -nitro-L-argininol which is further converted todipeptide-L-argininol by hydrogenation (on palladium on carbon Finally,the dipeptide-L-argininol is converted to the compound of the presentinvention, dipeptide-L-argininal, by oxidation of the alcohol moiety ofdipeptide-L-argininol to an aldehyde moiety using dichloroacetic acidand EDC in dimethylsulfoxide/toluene.

Other methods for the solution synthesis of peptide aldehydes have beenreported. For example, see McConnell et al., supra; at 87 and referencestherein; Bajusz et al., J. Med. Chem., 33:1729 (1990); Kawamura et al.,Chem. Pharm. Bull., 17:1902 (1969), and Someno et al., Chem. Pharm.Bull., 34:1748 (1986).

According to another aspect, the present invention is directed topharmaceutically acceptable salts of the compounds of formula (IA)."Pharmaceutically acceptable salt" includes within its definition saltsof the compounds of the present invention derived from the combinationof a such compounds and an organic or inorganic acid. In practice, theuse of the salt form amounts to use of the base form. The compounds ofthe present invention are useful in both free base and salt form, withboth forms being considered as being within the scope of the presentinvention. These salts include acid addition salts, for example, saltsof hydrochloric acid, hydrobromic acid, acetic acid, benzene sulfonicacid and other suitable acid addition salts.

3. Selection of Preferred Compounds

The compounds of the present invention are screened for their ability toinhibit thrombin or factor Xa and plasmin as set forth below. Certain ofthe preferred compounds are distinguished by their ability to inhibitthrombin, while not substantially inhibiting plasmin. With respect tothrombin and plasmin and as used herein, the term "not substantiallyinhibiting" means that the IC₅₀ (or K_(i)) for plasmin for a givencompound is greater than or equal to its IC₅₀ (or K_(i), respectively)for thrombin. Alternatively, certain of the preferred compounds aredistinguished by their ability to inhibit factor Xa, while notsubstantially inhibiting plasmin. With respect to factor Xa and plasminand as used herein, the term "not substantially inhibiting" means thatthe IC₅₀ (or K_(i)) for plasmin for a given compound is greater than orequal to its IC₅₀ (or K_(i), respectively) for factor Xa.

The compounds of the present invention are dissolved in buffer to givesolutions containing concentrations such that assay concentrations rangefrom 0 to 100 micromolar. In the assays for thrombin and plasmin, achromogenic synthetic substrate is added to a solution containing testcompound and the enzyme of interest and the residual catalytic activityof that enzyme is determined spectrophometrically. Likewise, in theassays for factor Xa and plasmin, a chromogenic synthetic substrate isadded to a solution containing test compound and the enzyme of interestand the residual catalytic activity of that enzyme is determinedspectrophometrically. The IC₅₀ of a compound of the present invention isdetermined from the rate of substrate turnover caused by the specificenzyme being measured. IC₅₀ is that concentration of test compoundgiving 50% inhibition of the rate of substrate turnover. Likewise, theK_(i) of a compound of the present invention is determined from the rateof substrate turnover caused by the specific enzyme being measured atvarious enzyme concentrations. K_(i) is that concentration of testcompound giving 50% inhibition of the rate of substrate turnover.Example A and B provides an exemplar of the in vitro assays used toselect the compounds of the present invention.

Preferred compounds of the present invention have an IC₅₀ (or K_(i)) of0.001 to 200 nM in the thrombin assay, and preferably the IC₅₀ (orK_(i)) for plasmin will not be less than the IC₅₀ (or K_(i)) forthrombin. More preferred are compounds having an IC₅₀ (or K_(i)) of0.001 to 100 nM in the thrombin assay. Especially preferred arecompounds having an IC₅₀ (or K_(i)) of about 0.001 to 20 nM in thethrombin assay and having a quotient of (IC₅₀ for plasmin)/(IC₅₀ forthrombin) [or (K_(i) for plasmin)/(K_(i) for thrombin)], of about 2 to100,000, preferably 10 to 100,000, and more preferably 100 to 100,000.

Alternatively, preferred compounds of the present invention have an IC₅₀(K_(i)) of 0.001 to 200 nM in the factor Xa assay, and preferably theIC₅₀ (K_(i)) for plasmin will not be less than the IC₅₀ (K_(i)) forfactor Xa. More preferred are compounds having an IC₅₀ (K_(i)) of 0.001to 100 nM in the factor Xa assay. Especially preferred are compoundshaving an IC₅₀ (K_(i)) of about 0.001 to 20 nM in the factor Xa assayand having a quotient of (IC₅₀ for plasmin)/(IC₅₀ for factor Xa) [or(K_(i) for plasmin)/(K_(i) for factor Xa)], of about 2 to 100,000,preferably 10 to 100,000, and more preferably 100 to 100,000.

4. Pharmaceutical Compositions

In another aspect, the present invention encompasses pharmaceuticalcompositions prepared for storage or administration which comprise atherapeutically effective amount of a compound of the present inventionin a pharmaceutically acceptable carrier.

The "therapeutically effective amount" of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy but will depend on such factors as weight, diet,concurrent medication and other factors which as noted those skilled inthe medical arts will recognize.

The "therapeutically effective amount" of the compound of the presentinvention can range broadly depending upon the desired affects and thetherapeutic indication. Typically, dosages will be between about 0.01mg/kg and 100 mg/kg body weight, preferably between about 0.01 and 10mg/kg, body weight.

"Pharmaceutically acceptable carriers" for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmeceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id.

The pharmaceutical compositions of the present invention may beformulated and used as tablets, capsules or elixers for oraladministration; suppositories for rectal administration; sterilesolutions and suspensions for injectable administration; and the like.The dose and method of administration can be tailored to achieve optimalefficacy but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize.

When administration is to be parenteral, such as intravenous on a dailybasis, injectable pharmaceutical compositions can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, or the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxilliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations(e.g., liposomes) may be utilized.

5. Utility and Methods

Compounds of the present invention when made and selected as disclosedare useful as potent inhibitors of thrombin in vivo and in vivo.Alternatively, compounds of the present invention when made and selectedas disclosed are useful as potent inhibitors of factor Xa in vitro andin vivo. As such, these compounds are useful as in vitro diagnosticreagents to prevent the clotting of blood and as in vivo pharmaceuticalagents to prevent thrombosis in mammals suspected of having a conditioncharacterized by abnormal thrombosis.

The compounds of the present invention are useful as in vitro diagnosticreagents for inhibiting clotting in blood drawing tubes. The use ofstoppered test tubes having a vacuum therein as a means to draw bloodobtained by venipuncture into the tube is well known in the medicalarts. Kasten, B. L., "Specimen Collection", Laboratory Test Handbood,2nd Edition, Lexi-Comp Inc., Cleveland pp. 16-17 (Edits. Jacobs, D. S.et al. 1990). Such vacuum tubes may be free of clot-inhibitingadditives, in which case, they are useful for the isolation of mammalianserum from the blood. They may alternatively contain clot-inhibitingadditives (such as heparin salts, EDTA salts, citrate salts or oxalatesalts), in which case, they are useful for the isolation of mammalianplasma from the blood. The compounds of the present invention are potentinhibitors of factor Xa or thrombin, and as such, can be incorporatedinto blood collection tubes to prevent clotting of the mammalian blooddrawn into them.

The compounds of the present invention are used alone, in combination ofother compounds of the present invention, or in combination with otherknown inhibitors of clotting, in the blood collection tubes. The amountto be added to such tubes is that amount sufficient to inhibit theformation of a clot when mammalian blood is drawn into the tube. Theaddition of the compounds to such tubes may be accomplished by methodswell known in the art, such as by introduction of a liquid compositionthereof, as a solid composition thereof, or liquid composition which islyophilized to a solid. The compounds of the present invention are addedto blood collection tubes in such amounts that, when combined with 2 to10 mL of mammalian blood, the concentration of such compounds will besufficient to inhibit clot formation. Typically, the requiredconcentration will be about 1 to 10,000 nM, with 10 to 1000 nM beingpreferred.

The compounds of the present invention are useful as a pharmaceuticalagent for preventing thrombosis in a mammal suspected of having acondition characterized by abnormal thrombosis.

Conditions characterized by abnormal thrombosis are well known in themedical arts and include those involving the arterial and venousvasculature of mammals. With respect to the coronary arterialvasculature, abnormal thrombosis (thrombus formation) characterizes therupture of an established atherosclerotic plaque which is the majorcause of acute myocardial infarction and unstable angina, as well asalso characterizing the occlusive coronary thrombus formation resultingfrom either thrombolytic therapy or percutaneous transluminal coronaryangioplasty (PTCA). With respect to the venous vasculature, abnormalthrombosis characterizes the condition observed in patients undergoingmajor surgery in the lower extremities or the abdominal area who oftensuffer from thrombus formation in the venous vasculature resulting inreduced blood flow to the affected extremity and a predisposition topulmonary embolism. Abnormal thrombosis further characterizesdisseminated intravascular coagulopathy which commonly occurs withinboth vascular systems during septic shock, certain viral infections andcancer, a condition wherein there is rapid consumption of coagulationfactors and systemic coagulation which results in the formation oflife-threatening thrombi occurring throughout the microvasculatureleading to widespread organ failure.

The present invention includes methods for preventing a condition in amammal suspected of having a condition characterized by abnormalthrombosis, comprising administering to said mammal a therapeuticallyeffective amount of a compound or a pharmaceutical composition of thepresent invention.

The compounds or pharmaceutical compositions of the present inventionare administered in vive, ordinarily in a mammal, preferably in a human.In employing them in vivo, the compounds or pharmaceutical compositionscan be administered to a mammal in a variety of ways, includingparenterally, intravenously, subcutaneously, intramuscularly,colonically, rectally, nasally or intraperitoneally, employing a varietyof dosage forms. Administration is preferably parenteral, such asintravenous on a daily basis.

In practicing the methods of the present invention, the compounds orpharmaceutical compositions of the present invention are administeredalone or in combination with one another, or in combination with othertherapeutic or in vivo diagnostic agents.

As is apparent to one skilled in the medical art, a therapeuticallyeffective amount of the compounds or pharmaceutical compositions of thepresent invention will vary depending upon the age, weight and mammalianspecies treated, the particular compounds employed, the particular modeof administration and the desired affects and the therapeuticindication. Because these factors and their relationship to determiningthis amount are well known in the medical arts, the determination oftherapeutically effective dosage levels, the amount necessary to achievethe desired result of preventing thrombosis, will be within the ambit ofone skilled in these arts. Typically, administration of the compounds orpharmaceutical composition of the present invention is commenced atlower dosage levels, with dosage levels being increased until thedesired effect of preventing in vivo thrombosis is achieved which woulddefine a therapeutically effective amount. For the compounds of thepresent invention, alone or as part of a pharmaceutical composition,such doses are between about 0.01 mg/kg and 100 mg/kg body weight,preferably between about 0.01 and 10 mg/kg, body weight.

To assist in understanding, the present invention will now be furtherillustrated by the following examples. These examples as they relate tothis invention should not, of course, be construed as specificallylimiting the invention and such variations of the invention, now knownor later developed, which would be within the purview of one skilled inthe art are considered to fall within the scope of the invention asdescribed herein and hereinafter claimed.

EXAMPLES Example 1 Preparation of alpha-N-t-butoxycarbonyl-N^(g)-nitroargininal ##STR54## A. Procedure 1:

The following procedure for the synthesis ofalpha-t-butoxycarbonyl-N^(g) -nitro-argininal, the title compound, is anexample of a general procedure for the preparation of Boc-amino acidaldehydes, see Patel et al., Biochim. Biophys. Acta, 748, 321-330(1983).

In 200 mL dry THF, 12.7 g Boc-N^(g) -nitro-arginine (40 mmoles) and 7.0g carbonyldiimidazole (CDI; 43 mmoles) were added at room temperatureand allowed to stir for 30 minutes. The reaction mixture was cooled to-78° C. and 35 mL of a solution of LiAlH₄ (1M in THF) were addeddropwise over thirty minutes. The reaction was allowed to stir for anadditional hour at -78° C. Next, 18 mL of acetone were added and thismixture was quickly added to 400 mL of 1N HCl. The mixture was extractedtwice with 100 mL of ethyl acetate. The ethyl acetate washes werecombined and then washed two times each with 100 mL water, 100 mLsaturated NaHCO₃ and 100 mL saturated NaCl. The solution was dried(MgSO₄) and concentrated to a foam. The crude weight of the titlecompound was 6.36 g (21 mmole; yield 52%

B. Procedure 2:

Alternatively, the title compound was synthesized by a modification ofthe procedure of Fehrentz, J. A. and Castro, B., Synthesis, 676 (1983).

11.4 mL of N-methyl piperidine was slowly added to a stirred suspensionof 8.42 g (94 mmole) of N,O-dimethylhydroxylamine hydrochloride in 75 mLdichloromethane which had been cooled to about 0° C. The solution wasallowed to stir for 20 minutes which gave the free hydroxylamine, thenwas kept cold for use in the next step.

In a separate flask, 30.0 g (94 mmole) of Boc-N^(g) -nitroarginine wasdissolved by heating in about 1400 mL of THF, then the mixture wascooled under nitrogen to 0° C. 11.4 mL of N-methylpiperidine and 12.14mL (94 mmole) of isobutylchloroformate was added and the mixture stirredfor 10 minutes. The free hydroxylamine prepared above was added all atonce and the reaction mixture was allowed to warm to room temperature,then stirred overnight.

The resulting precipitate was filtered off, then washed with 200 mL ofTHF. After concentrating the filtrates to about 150 mL under vacuum, 200mL of ethyl acetate was added, followed by ice to cool the solution. Thecooled ethyl acetate phase was washed with two 75 mL portions of 0.2Nhydrochloric acid, two 75 mL portions of 0.5N sodium hydroxide, oneportion of 75 mL of brine, then the organic phase was dried over MgSO₄.Upon concentration in vacuum, 22.7 g (70% yield) of solid Boc-N^(g)-nitroarginine N-methyl-O-methylcarboxamide was recovered. Thin layerchromatographic analysis in 9:1 dichloromethane/methanol (silica gel)showed one spot.

A flask was placed under a nitrogen atmosphere and cooled to -50° C.,then charged with 70 mL (70 mmole) of 1N lithium aluminum hydride (inTHF) and 500 mL of dry THF. 50 mL of a solution containing 66 mmole ofBoc-N^(g) -nitroarginine N-methyl-O-methylcarboxamide in dry THF wasslowly added while the temperature of the reaction mixture wasmaintained at -50° C. After allowing the reaction mixture to warm to 0°C. by removal of the cooling, it was recooled to -30° C., at whichtemperature, 100 mL (0.2 mole) of 2N potassium bisulfate was added withstirring over about a 10 to 15 minute period. The reaction mixture wasthen allowed to stir at room temperature for 2 hours. After filteringoff the precipitate, the filtrate was concentrated to 100 mL undervacuum. The concentrate was poured into 800 mL ethyl acetate, then wassuccessively washed with two 50 mL portions of 1N hydrochloric acid, two50 mL portions of saturated sodium bicarbonate, one 50 mL portion ofbrine. The combined aqueous extracts were extracted with 3-100 mLportions of ethyl acetate. All of the ethyl acetate washes werecombined, then was dried over MgSO₄. The mixture was concentrated undervacuum to yield 18.5 g (95%) of the title compound.

Example 2 Preparation of trans-4-(aminomethyl)-cyclohexane carboxylicacid benzyl ester para-toluenesulfonate salt ##STR55##

50 g (0.318 moles) of trans-4-(aminomethyl)-cyclohexane carboxylic acid,61.7 g (0.324 moles) p-toluenesulfonic acid, 250 mL (2.4 moles) benzylalcohol and 250 mL toluene were combined and stirred. The mixture wasrefluxed for 24 hours and the liberated water was removed azeotropicallyby means of a Dean-Stark apparatus. A clear solution was obtained after5 hours of refluxing. The solution was allowed to cool to roomtemperature and the product crystallized. The mixture was vacuumfiltered, washed with ether and dried in a vacuum oven to give 128.12 g(96% yield.). Reference: Greenstein, Jesse P.; Winitz, Milton. Chemistryof the Amino Acids. vol. 2, (1986), 942. ¹ H NMR (CD₃ OD) δ1.05 (m, 2H),1.43 (m, 2H), 1.59 (m, 1H), 1.85 (m, 2H), 2.03 (m, 2H), 2.33 (m, 1H),2.35 (s, 3H), 2.75 (d, 2H), 5.09 (s, 2H), 7.23 (d, 2H), 7.32 (m, 5H),7.69 (d, 2H). M.P. 154°-156 C.

Example 3 Preparation of1-t-butoxycarbonyl-semicarbazidyl-trans-4-methyl cyclohexane carboxylicacid benzyl ester ##STR56##

3.24 g (0.02 moles) carbonyldiimidazole (CDI) were dissolved in 45 mL ofdimethylformamide (DMF) at room temperature under nitrogen. A solutionof 2.48 g (0.02 moles) t-butyl carbazate in 45 mL DMF was addeddropwise. Next 8.38 g (0.02 moles) of solid benzyl ester of Example 2was added, followed by the dropwise addition of 3.06 mL of triethylamine(TEA) over a 30 minute period. The reaction was allowed to stir at roomtemperature under nitrogen for one hour. Water (100 mL) was added andthis mixture was extracted three times with 50 mL of ethyl acetate. Theethyl acetate layers were combined and extracted two times each with 75mL 1N HCl H₂ O, NaHCO₃, NaCl and dried with MgSO₄. The mixture wasfiltered and the solution was concentrated to give an oil. This materialcould be purified by recrystallization from ethyl acetate/hexanes(M.P.=106°-108° C.) or used directly in the next step. ¹ H NMR (CDCl₃)δ0.94 (m, 2H), 1.42 (m, 2H), 1.45 (s, 9H), 1.81 (m, 2H), 2.02 (m, 2H),2.27 (m, 1H), 3.17 (t, 2H), 5.09 (s, 2H), 5.51 (t, 1H), 6.46 (s, 2H),7.34 (m, 4H).

Example 4 Preparation of1-(t-butoxycarbonyl)-3-semicarbazidyl-trans-4-methyl-cyclohexanecarboxylic acid ##STR57##

To the crude Boc-benzyl ester of Example 3, 250 mL of methanol (MeOH)and 500 mg of 10% palladium on carbon were added. After shaking on thehydrogenator for one hour at 5 psi, the mixture was filtered with Celitethrough a fine fritted filter. The solution was concentrated to a foam,dichloromethane was added and a precipitate formed. The mixture was kept5° C. for 65 hours. The crystallized material was filtered with etherand 4.0 g of crude product were obtained (12.7 mmoles; yield 62% overallyield from the compound of Example 2) ¹ H NMR (CD₃ OD), δ0.96, (m, 2H),1.42 (m, 2H), 1.46 (s, 9H), 1.82 (m, 2H), 1.97 (m, 2H), 2.18 (m, 1H),3.0 (t, 2H). M.P.=185°-189° C.

Example 5 Preparation of semicarbazidyl-trans-4-methyl cyclohexanecarboxylic acid trifluoroacetate salt ##STR58##

315 mg (1 mmole) of compound of Example 4 was added to 10 mL oftrifluoroacetic acid (TFA) at 0° C. and the resulting solution wasallowed to stir for 30 minutes. After this time the solution was addeddropwise to 75 mL of diethyl ether. A precipitate formed, and themixture was filtered and washed with diethyl ether. Weight of crudeproduct was 254 mg, 0.77 mmoles; yield (77%). ¹ H NMR (CD₃ OD), δ1.0 (m,2H), 1.38 (m, 2H), 1.43 (m, 1H), 1.84 (m, 2H), 2.01 (m, 2H), 2.22 (m,1H), 3.04 (d, 2H). M.P.=154°-156° C.

Example 6 Preparation of alpha-(t-butoxycarbonyl)-N^(g) -nitroargininal-semicarbazonyl-trans-4-methyl-cyclohexane carboxylic acid##STR59##

A solution of 13.7 g (41.6 mmoles) of the compound of Example 5, 18.0 g(˜59 mmoles) of crude compound of Example 1 in 135 mL ethanol containing45 mL of water, was treated with 9.41 g (69 mmoles) of sodium acetate(NaOAc) and refluxed for one hour. This solution was allowed to cool andthen poured into 0.1N HCl and extracted three times with ethyl acetate.The combined organic phase was washed with water, then brine, dried(MgSO₄) and concentrated to a small volume. This cloudy mixture wasallowed to set overnight at 5° C. to precipitate the product, which wasisolated by filtration and dried under vacuum. This gave 9.9 g, 47%yield based on the amount of the compound of Example 5 used. ¹ H NMR(CD₃ OD) δ1.0 (m, 2H), 1.43 (s, 9H), 1.45-2.20 (m, 13H), 3.09 (d, 2H),3.30 (m, 2H), 4.18 (bs, 1H), 7.10 (d, 1H). M.P.=162°-163° C.

Example 7 Synthesis of Semicarbazone Solid Phase ##STR60##

The title resin, a solid phase reagent, was prepared by coupling of thecompound of Example 6 to methyl-benzhydralamine (MBHA) resin as follows.

MBHA resin (0.8 g, 0.5 mmoles amino groups) was placed in a reactionvessel and washed successively one time with DCM, three times with DMF,two times with 10% DIEA/DMF, and four times with DMF. Each wash is doneby adding 10 mL of solvent to the resin, agitating the mixture for 1-2minutes, and then draining the solvent.

The washed MBHA resin was placed in a reaction vessel and 5 mL of DMFwas added. To this mixture, NMM (0.102 mL, 1 mmole), BOP (443 mg, 1mmole) and the compound of Example 6 (500 mg, 1 mmole) was added. Themixture was then mixed on a rotating wheel for 16 hours. After thistime, the resin was filtered off and washed successively three timeswith DMF, two times with 10% DIEA/DMF and three times with DMF. Theresin was then washed successively one time with DCM, one time withmethanol and one time with diethyl ether to yield the title resin. Eachwash was again done by adding 10 mL of solvent to the resin, agitatingthe mixture for 1-2 minutes, and then draining the solvent.

The title resin showed a 98-99% coupling yield by ninhydrin. The yieldwas calculated on the basis of the mmoles/g of amino groups on thestarting MBHA resin.

Example 8 Solid Phase Synthesis

The compounds of the present invention may be made by the solid-phasesynthesis method as described in this example. This synthesis method iscarried out in three stages--coupling, cleavage and hydrolysis.

Washing of the resin is common step in this procedure. Accordingly,unless otherwise specified, a wash is defined as the adding 5-10 mL of aspecified solvent to the resin, agitating the mixture for 1-2 minutes,and then draining the solvent away from the resin.

A. Coupling.

In the first stage of the present method, specified intermediates arecoupled to the resin of Example 7 in the following manner:

1. The resin of Example 7 (0.5 g, 0.25 mmole alpha-amino groups) isplaced in a reaction vessel and washed three times with DCM.

2. The alpha-amino groups of the resin are deprotected by twiceimmersing the resin in 5-10 mL of 50% TFA in DCM, with first time for 5minutes and the second time for 30 minutes. The resin is then washed twotimes with DMF, two times with DCM, and two times with DMF.

3. The deprotected resin is neutralized by immersing the resin in 5-10mL of 5% DIEA in DMF for 7 minutes. The resin is then washed two timeswith DMF, two times with DCM, and two times with DMF.

4. The deprotected resin is combined with 5 mL of DMF and then 1.0 mmoleof amino acid, amino acid analog, peptide or peptide analog (which has afree C-terminal carboxy group, is protected by a Boc group or otherwiseblocked at the N-terminal amino group, and suitably protected at allother reactive functionalities), TBTU (0.321 mg, 1.0 mmole), HOBt (0.135mg, 1.0 mmole), NMM (0.11 mL, 1.0 mmole) and 3 mL of DMF, then themixture is agitated for three hours. BOP (0.442 mg, 1.0 mmole) may besubstituted for the TBTU.

5. If further amino acids, amino acid analogs, peptides or peptideanalogs are to be sequentially coupled, steps 2-4 is repeated until thedesired compound is achieved.

6. The resin is separated from the DMF solution and is washed threetimes with DMF, three times with DCM, three times with methanol, andthree times with diethyl ether. The resin is then dried under vacuum.

B. Cleavage.

In the second stage of the present method, the desired compound in theform of a semicarbazone intermediate is cleaved from the resin.

The dried resin (0.50 g) from Step A is placed in a reaction vessel andanisole (0.5 mL) is added. Thiocresol (0.1 mL) is additionally added forthe cleavage of certain specified compounds of the present invention.After cooling the reaction vessel to -20° C., gaseous hydrofluoric acid(12.0 mL) is distilled into the reaction mixture with stirring.

After stirring for 30 minutes at -20° C., the reaction mixture is warmedto -10° C. and stirred for an additional 2 hours at this temperature.After this time, the reaction mixture is warmed to 0° C. and thehydrofluoric acid is distilled off with a stream of N₂. The resin isthen washed twice with diethyl ether.

The title compound is extracted from the resin by successively washingthe resin with 0.1M ammonium bicarbonate (50 mL) and water (100 mL),which are then combined and extracted three times with diethyl etherusing 25 mL of the solvent for each extraction. The extracted aqueoussolution is frozen and lyophilized to give the crude semicarbazone oftitle product. C. Hydrolysis.

In the third stage of the present method, the semicarbazone intermediateis hydrolyzed to the compound of the present invention.

Crude semicarbazone (0.25 mmole) from Step B is placed in a reactionvessel, followed by 6 mL acetic acid, 12 mL THF and 6 mL of water(adjusted to pH 1 with trifluoroacetic acid). Stirring is commenced and1 mL of ethyl acetoacetate is added. Additional 1 mL portions of ethylacetoacetate are added every 1-8 hours until the semicarbazone ishydrolyzed to the title compound. The reaction mixture is then reducedto dryness under vacuum to give crude title compound.

The crude title compound is purified by high pressure liquidchromatography, using a reverse-phase column containing a C-18 resincomprised of 10 micron-size gel particles with a 300 angstrom pore size.The column is eluted with a water-acetonitrile (both containing 0.1%trifluoroacetic acid) gradient, where the gradient is run from 5% to 40%acetonitrile. The column fractions are analyzed by analytical highpressure liquid chromatography using a reverse phase C-18 column and thesame gradient system. The fractions containing pure product are pooled,then lyophilized to yield the title product.

Example 9 Preparation of L-methionine sulfone-L-proline-O-benzyl esterhydrochloride salt ##STR61## A. Procedure 1:

N-Boc-L-methionine sulfone-L-proline-O-benzyl ester was prepared byadding to a solution of N-Boc-L-methioninesulfone (14.0 g, 50.0 mmole)in dichloromethane (150 mL) at 0° C., HOBt (10.1 g, 75 mmole) followedby DCC (11.33 g, 55.0 mmole). The mixture was stirred for 10 minutes,and then proline benzyl ester hydrochloride salt (50.0 mmole, 12.0 g)was added followed by NMM (100 mmole, 10.9 mL). The resulting mixturewas stirred in an ice bath and allowed to come to room temperature over12 hours. The mixture was filtered to remove dicyclohexylurea and ethylacetate (300 mL) is added. The organic phase was then added to aseparatory funnel and washed with saturated aqueous sodium bicarbonate,brine and then 1M aqueous HCl. The organic phase was dried overmagnesium sulfate and then filtered. The organic phase was then reducedon a rotary evaporator under vacuum and then on a high vacuum line toremove traces of solvent to provide 23.5 g of a white solid (100%).Rf=0.34 (silica, 5:95 methanol/chloroform).

To a solution of N-Boc-L-methionine sulfone-L-proline-O-benzyl ester(23.5 g, 50 mmole) in dry dioxane (300 mL) was added 100 mL of a 4MHCl/dioxane solution. The mixture was then stirred at room temperaturefor 1 hour until the starting material disappeared as shown by thinlayer chromatography analysis (silica, 10% chloroform in methanol).Diethyl ether was added to the mixture to precipitate the title compoundas a white hydrochloride salt. The mixture was filtered on a Buchnerfunnel and the solid was then dried under high vacuum to give 20.16 g(100%) of the title compound as a white solid.

B. Procedure 2:

Alternatively, the title compound was synthesized by the followingmethod.

To a solution of N-Boc-L-methionine sulfone (5 g, 20 mmole) in 80 mL ofdry DMF was added L-proline-O-benzyl ester hydrochloride salt (4.8 g, 20mmole) followed by BOP (8.9 g, 20 mmole) and NMM (5.5 mL, 20 mmole). Themixture was stirred for 16 hours at room temperature. The reactionmixture was dissolved in 600 mL of ethyl acetate and washed with 200 mLeach of water, 1M aqueous HCl water, saturated aqueous sodiumbicarbonate and brine. The organic phase was dried over magnesiumsulfate, filtered and the solvent removed under vacuum to yield an oil.

To the solution of the resulting oil in 20 mL dichloromethane, 100 mL ofa 4M solution of HCl in dioxane was added. After stirring for 16 hours,the solvent was removed under vacuum. The resulting oil was precipitatedusing diethyl ether, filtered and dried under vacuum to provide 7.49 g(86% yield) of the title compound as a white solid. Thin layerchromatography analysis of the title compound showed a single spot withRf=0.1 (silica, 1:9 methanol/dichloromethane).

Example 10 Preparation ofN-(1-butanesulfonyl)-L-methioninesulfone-L-proline ##STR62##

A 6.0 g (12.4 mmole) of the compound of Example 9 was reacted with 2.07mL (16 mmole) of 1-butanesulfonylchloride and 5.0 mL (36 mmole) oftriethylamine in dichloromethane from 0° C. to room temperature. Thereaction mixture was poured into saturated aqueous bicarbonate andextracted with ethyl acetate (2×100 mL). The organic phase was washedwith brine and 1M aqueous HCl. The organic phase was separated and driedover magnesium sulfate, filtered and reduced under vacuum to give 5.73 gof a viscous oil.

The oil was mixed with 2M potassium hydroxide (20 mL) and 100 mLmethanol at room temperature for two hours. The methanol was reducedunder vacuum and the aqueous solution was then washed with ether (2×50mL) and then neutralized with 1M HCl to a pH of 1. The aqueous solutionwas then extracted with ethyl acetate (2×100 mL) and dried over MgSO₄,filtered and reduced under vacuum to give 2.85 g of the title compoundas a viscous foamy solid. The overall yield was 60.5%.

Example 11 Preparation ofN-(1-butanesulfonyl)-L-methioninesulfone-L-proline-L-argininal ##STR63##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 10 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 538.2.

Example 12 Preparation of cyclohexylmethylsulfonate sodium salt##STR64##

To a solution of sodium bisulfite (32 g, 307 mmole) in H₂ O (200 mL) wasadded cyclohexylmethyl bromide (21 mL, 150 mmole). The mixture was thenstirred vigorously while heated at reflux for 48 hours. Upon cooling toroom temperature, a white precipitate formed. The precipitate wascollected on a Buchner funnel and washed with diethyl ether to wash awayresidual cyclohexylmethyl bromide. The white plate-like solid was driedunder high vacuum overnight to provide 11.8 g (39%) of the titlecompound which had a m.p. >400° C.

Example 13 Preparation of cyclohexylmethyl sulfonyl chloride ##STR65##

To the compound of Example 12 (4.0 g, 20 mmole) was added POCl₃ (15 mL)and the mixture heated at 100° C. for 48 hours. Upon cooling to roomtemperature the mixture was poured onto crushed ice and then the aqueousphase was extracted with ethyl acetate (2×100 mL). The combined organicextracts were then washed with saturated aqueous sodium bicarbonate(2×50 mL), brine and then dried over MgSO₄, filtered and the solvent wasevaporated under vacuum to provide 3.46 g of the title compound as alight yellow oil. Thin layer chromatography analysis of the titlecompound showed a single spot with R_(f) =0.53 (silica, 4:1hexanes/ethyl acetate).

Example 14 Preparation of N-Boc-L-methionine sulfone-O-benzyl ester##STR66##

To a solution of N-Boc-L-methionine sulfone (50 g, 178 mmole) in dry THF(500 mL) which had been chilled to 0° C., carbonyl diimidazole (34.6 g,214 mmole) was added in small portions. After 30 minutes, the mixturewas warmed to room temperature for 2 hours until all of the CO₂evolution ceased. After this time, benzyl alcohol (27.6 mL, 267 mmole)was added and the reaction stirred for 12 hours.

The reaction mixture was then reduced in volume under vacuum and theresulting residue was diluted with ethyl acetate (500 mL). The organicphase was then washed with saturated bicarbonate (1×100 mL), brine (100mL), then saturated aqueous citric acid (1×100 mL), dried over MgSO₄,filtered and the solvent removed under vacuum to provide a white solid.The white solid was washed with a 1:1 mixture of diethyl ether/hexanes(300 mL) and filtered off on a Buchner funnel to provide 50.0g (92%) ofthe title compound. Thin layer chromatography analysis of the titlecompound showed a single spot with R_(f) =0.18 (silica, 3:2hexanes/ethyl acetate).

Example 15 Preparation of L-methionine sulfone-O-benzyl esterhydrochloride salt ##STR67##

To the compound of Example 14 (50.0g), 200 mL of a 4M solution of HCl indioxine was added. The solid eventually dissolved over 2 hours andshowed no starting material by thin layer chromatography. The solutionwas then reduced in volume under vacuum and the resulting solid waswashed with diethyl ether to provide 55.0 g (100%) of the title compoundas a white solid.

Example 16 Preparation of N-cyclohexylmethanesulfonyl-L-methioninesulfone-O-benzyl ester ##STR68##

To a suspension of the compound of Example 15 (4.6 g, 15 mmole) in dryCH₃ CN (35 mL) cooled to 0° C. in an ice bath, the compound of Example13 (N-cyclohexylmethane sulfonyl chloride, 3.46 g, 17.6 mmole) as asolution in CH₃ CN (10 mL) was added, followed by pyridine (3.8 mL, 45mmole). The reaction was allowed to stir in the ice bath for 15 hourswhile slowly warming to room temperature. The solvent was evaporatedunder vacuum to give a residue.

The resulting residue was dissolved in ethyl acetate (200 mL) and thesolution was washed with saturated aqueous bicarbonate (50 mL), brine(50 mL), saturated aqueous citric acid (50 mL), and dried over MgSO₄.The solution was filtered and evaporated under vacuum to provide 3.3 gof a yellow oil. This crude product was purified by silica gel flashchromatography to provide 1.63 g (25%) of the title compound as a clearviscous oil. Thin layer chromatography analysis of the title compoundshowed a single spot with R_(f) =0.29 (silica, 3:2 ethylacetate/hexanes).

Example 17 Preparation of N-cyclohexylmethanesulfonyl-L-methioninesulfone ##STR69##

To a solution of the compound of Example 16 (1.6 g, 3.7 mmole) in CH₃ OH(50 mL), 250 mg 10% palladium on carbon was added. This mixture was thensubjected to atmospheric hydrogenation at room temperature for 12 hours.The reaction mixture was then filtered through a pad of celite and thesolvent was evaporated under vacuum to provide the title compound (1.06g, 88%) as a white solid. Thin layer chromatography analysis of thetitle compound showed a single spot with R_(f) =0.2 (silica, 9:1chloroform/methanol).

Example 18 Preparation of N-cyclohexylmethanesulfonyl-L-methioninesulfone-L-proline-L-argininal ##STR70##

The title compound was prepared in the same manner as described inExample 8.

N-Boc-L-proline was first attached to the resin of Example 7, followedby the compound of Example 17. After cleavage of the title compound as aprotected semicarbazone from the resin, the semicarbazone was hydrolyzedto give the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 578.2.

Example 19 Preparation of N-(1-naphthylenesulfonyl)-L-methioninesulfone-L-proline-O-benzyl ester ##STR71##

To a solution of the compound of Example 9 (3 g, 6.9 mmole) in 70 mL ofdry acetonitrile, 1-naphthylenesulfonyl chloride (2.3 g, 10.3 mmole) wasadded followed by dry pyridine (4.1 mL, 34.4 mmole). After stirring for16 hours at room temperature, the solvent was removed under vacuum andthe resulting oil was dissolved in 500 mL ethyl acetate and washed with100 mL each of water, 1M aqueous HCl water, saturated aqueous sodiumbicarbonate and brine. The organic phase was dried over MgSO₄, filteredand the solvent removed under vacuum to yield an oil.

The oil was filtered down a pad of silica (50 g), rinsing with two 500mL portions of dichloromethane followed by three 500 mL portions of 1:9methanol/dichloromethane. The appropriate fractions were concentratedunder vacuum to provide 3.8 g (99% yield) of the title compound. Thinlayer chromatography analysis of the title compound showed a single spotwith Rf=0.5 (silica, 1:9 methanol/dichloromethane). Example 20

Preparation of N-(1-naphthylenesulfonyl)-L-methionine sulfone-L-proline##STR72##

To a solution of the compound of Example 19 (3.8 g, 6.8 mmole) in 10 mLof THF and 250 mL of methanol under a nitrogen blanket, 10% palladium oncarbon (2 g) was added. This mixture was hydrogenated at 1 atmospherefor 16 hours. The mixture was then filtered and concentrated undervacuum to provide 3 g (96% yield) of the title compound as a whitesolid. Thin layer chromatography analysis of the title compound showed asingle spot with Rf=0.2 (1:9 methanol/dichloromethane).

Example 21 Preparation of N-(1-naphthylenesulfonyl)-L-methioninesulfone-L-proline-L-argininal ##STR73##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 20 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 608.2.

Example 22 Preparation of N-(2-naphthylenesulfonyl)-L-methioninesulfone-L-proline-O-benzyl ester ##STR74##

A 3 g (6.878 mmole) portion of the compound of Example 9 was added to 69mL of acetonitrile. To this mixture, 2.34 g (10.3 mmole, 1.5 eq.) of2-naphthylenesulfonylchloride and 4.20 g (4.12 mL, 34.4 mmole, 5 eq.) ofpyridine was added and the mixture was stirred for 10 hours. The mixturewas then concentrated under vacuum, diluted with ethyl acetate (500 mL)and washed with 1M HCl water, aqueous sodium bicarbonate, and brine. Theorganic phase was dried over MgSO₄ and concentrated under vacuum. Thinlayer chromatography (silica, 10% methanol in dichloromethane) showedsome 2-naphthylsulfonylchloride. The mixture was then filtered down aplug of silica, eluting with dichloromethane (100 mL), then 10%methanol/dichloromethane (200 mL). Removal of the solvent under vacuumfrom the collected fractions gave 3.96 g of the title compound.

Example 23 Preparation of N-(2-naphthylenesulfonyl)-L-methioninesulfone-L-proline ##STR75##

A 3.96 g (7.088 mmole) portion of the compound of Example 22 wasdissolved in 250 mL of methanol with a trace of THF. To this solution, 2g of 10% palladium on carbon was added under a nitrogen blanket. Themixture was then stirred under hydrogen at one atmosphere of pressurefor 10 hours. After this time, thin layer chromatography (silica, 10%methanol in dichloromethane) showed no starting material. This solutionwas then filtered through a nylon filter and concentrated under vacuumto give 3.2 g (96%) of the title compound.

Example 24 Preparation of N-(2-naphthylenesulfonyl)-L-methioninesulfone-L-proline-L-argininal ##STR76##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 23 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 608.2.

Example 25 Preparation of N-benzylsulfonyl-L-methioninesulfone-L-proline-O-benzyl ester ##STR77##

To a solution of the compound of Example 9 (20.0 mmole, 8.08 g) in dryacetonitrile (100 mL) cooled to 0° C., alpha-toluenesulfonylchloride(20.0 mmole, 3.8 g) was added all at once followed by pyridine (50.0mmole, 4.2 mL). The mixture was then stirred in the ice bath for 12hours eventually warming to room temperature.

The reaction mixture was reduced to a residue under vacuum. The residuewas taken up ethyl acetate (300 mL) and washed with saturated aqueoussodium bicarbonate, brine, 1M aqueous HCl (100 mL), dried over MgSO₄,filtered and evaporated under vacuum to provide 8.8 g (100%) of thetitle compound as a foamy golden solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.31 (silicagel, 95:5 chloroform/methanol). The solid was filtered through a plug ofsilica gel (50 g) using ethyl acetate as eluent before hydrogenation toeliminate possible sulfur related impurities.

Example 26 Preparation of N-benzylsulfonyl-L-methioninesulfone-L-proline ##STR78##

To a solution of the compound of Example 25 (8.8 g, mmole) in methanol(300 mL), 1.0 g of 10% palladium on carbon was added. The mixture wasthen hydrogenated at 1 atmosphere of hydrogen gas and room temperaturefor 12 hours with stirring. The mixture was then filtered and theorganic phase reduced under vacuum to provide 8.0 g (100%) of the titlecompound as a white foamy solid.

Example 27 Preparation of N-benzylsulfonyl-L-methioninesulfone-L-proline-L-argininal ##STR79##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 26 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 572.2.

Example 28 Preparation of N-Boc-N^(g) -nitro-argininol. ##STR80##

To a suspension of Boc-N^(g) -nitro-arginine (370 g, 1.15 moles) in 6liters of dry THF at -78° C., borane.THF complex 1.0M (2.6 liters) wasslowly added. The reaction temperature was controlled so that it did notexceed -60° C. After the addition was complete, the reaction was placedin a freezer at -20° C. overnight.

The following day, the greenish-yellow reaction mixture was cooled to-78° C. and slowly quenched with 3 liters of anhydrous methanol. Twohours after this quenching, the mixture was warmed to 25° C. and stirredfor an additional 2 hours. The solvent is removed under vacuum to yieldthe title compound (360 g). This intermediate is used as is in couplingreactions and the major product had a Rf=0.28 (silica, 90:10dichloromethane/methanol) by thin layer chromatography.

Example 29 Preparation of N-(2-propylpentanoyl)-L-methioninesulfone-L-proline-O-benzyl ester ##STR81##

To a solution at 15° C. containing the compound of Example 9 (88.06 g,0.178 mole), 2-propylpentanoic acid (33.45 mL, 0.2136 mole), BOP (94.5g, 0.2136 mole) and 600 mL of dry DMF, NMM (117 mL, 1.068 mole) wasslowly added. The reaction mixture was warmed slowly to 25° C. and thenwas stirred overnight.

On the following day, 1 liter of water was added to the reaction mixtureand it was extracted 3 times with 500 mL of ethyl acetate. The organicextracts were combined and washed with 3-300 mL portions of 1.0N HCl,3-300 mL portions of saturated sodium bicarbonate and 1-100 mL portionof brine. The organic phase was then dried over magnesium sulfate,filtered, and then the solvent was removed under vacuum to yield 84.6 g(96 %) of the title compound. Thin layer chromatography analysis of thetitle compound showed a single spot with Rf=0.35 (silica gel, 95:5dichloromethane/methanol).

Example 30 Preparation of N-(2-propylpentanoyl)-L-methioninesulfone-L-proline ##STR82##

To a solution of the compound of Example 29 (37 g, 0.07495 mole) and 500mL methanol, 3.7 g 10% palladium on carbon wet with dichloromethane wasadded. The mixture was hydrogenated in a Parr Hydrogenator at 25 psi for7 hours, after which time the mixture was filtered and the solventremoved under vacuum to yield 29.71 g (98%) of the title compound as awhite solid. Thin layer chromatography analysis of the title compoundshowed a single spot with Rf=0.15 (silica, 85:15dichloromethane/methanol).

Example 31 Preparation of N-(2-propylpentanoyl)-L-methioninesulfone-L-proline-N^(g) -nitro-L-argininol ##STR83##

To a solution of compound of Example 28 (34 g, 0.1117 mole) in 500 mL ofmethanol at 0° C., 1.2 liters of saturated HCl/ methanol solution wasadded. After 30 minutes, the ice bath was removed and the reactionmixture was allowed to stir for 2 hours. After this time, the solventwas removed under vacuum and the resulting solid was used as is withoutfurther purification.

The resulting solid was dissolved in 1 liter of acetonitrile. To thissolution, the compound of Example 30 (49.56 g, 0.1225 mole) and BOP(59.11 g, 0.1337 mole) was added, followed by the slow addition of NMM(73.5 mL, 0.668 mole). The mixture was stirred overnight at roomtemperature. After this time, the solvent was removed under vacuum andthe residue was chromatographed on silica gel eluting with a 100:0 to90:10 gradient of dichloromethane and methanol. 40.5 g of the titlecompound as a white solid was isolated by evaporation of solvents fromthe collected fractions. Thin layer chromatography analysis of the titlecompound showed a single spot with Rf=0.38 (silica gel, 95:5dichloromethane/methanol).

Example 32 Preparation of N-(2-propylpentanoyl)-L-methioninesulfone-L-proline-L-argininol ##STR84##

To a solution of the compound of Example 31 (6 g, 0.0101 mole), 3 mL ofacetic acid and 100 mL methanol, 0.6 g 10% palladium on carbon wet withdichloromethane was added. This mixture was hydrogenated in a ParrHydrogenator at 45 psi for 16 hours. After this time, the mixture wasfiltered and the organic phase was removed under vacuum to yield 4.7 g(77%) of the title compound as a glass. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.17 (silicagel, 85:15 dichloromethane/methanol).

Example 33 Preparation of N-(2-propylpentanoyl)-L-methioninesulfone-L-proline-L-argininal ##STR85##

To a solution of the compound of Example 32 (5.4 g, 9.9 mmole),dichloroacetic acid (4.07 mL, 49.5 mmole), 75 mL of dimethyl sulfoxideand 75 mL of toluene at 0° C., EDC (18.93 g, 99 mmole) was added slowly.The ice bath was removed after 5 minutes and the reaction was allowed towarm to room temperature for 1.5 hours. After this time, the toluene wasremoved under vacuum and the resulting solution was diluted with 750 mLof HPLC-grade water and filtered. This solution was then chromatographedusing a C-18 reverse phase column using a 30 minute gradient comprising100:0 to 60:40 of HPLC-grade water/acetonitrile containing 0.1%trifluoroacetic acid. The fractions were combined and lyophilyzed togive 4.47 g (83.5%) of the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 544.3.

Example 34 Preparation of S-methyl-L-cysteine ##STR86##

A 1.49 liter aqueous solution of commercially available (Aldrich)L-cysteine hydrochloride monohydrate (50.0 g, 284.5 mmole) and bariumhydroxide octahydrate (94.24 g, 298.7 mmole), at room temperature, wastreated with dimethyl sulfate (37.32 g, 295.9 mmole) added dropwise over40 minutes. After stirring an additional 2 hours, concentrated sulfuricacid (29.27 g, 298.7 mmole) dissolved in 100 mL of de-ionized water wasadded in portions. The slurry was filtered and the filtrate concentratedto 70 mL under vacuum at 40° C. The solution was adjusted to pH 5-6 (pHpapers) with concentrated ammonium hydroxide causing a precipitate toform. The slurry (140 mL aqueous volume) was diluted with 240 mL ofethanol, stirred and cooled at 3° C. The white solid was filtered,washed with cold 2:1 ethanol/water (50 mL) and dried under high vacuumat room temperature to give 33.3 g (86.6% yield) of the title compound.

Example 35 Preparation of N-Boc-S-methyl-L-cysteine ##STR87##

The compound of Example 34 (15.0 g, 110 mmole) was suspended in 130 mLof saturated sodium bicarbonate (approximately 1.1M). A solution ofdi-t-butyl dicarbonate (26.4 g, 121 mmole) in 60 mL of THF was added andvigorously stirred for 18 hours at room temperature.

The reaction mixture was extracted with diethyl ether (100 mL). Theresulting aqueous phase was layered with ethyl acetate (200 mL) andacidified to pH 2 (pH papers) with 1N hydrochloric. The aqueous layerwas further extracted with ethyl acetate (3×200 mL). The organicextracts were combined, washed with brine, dried with MgSO₄ and thesolvent evaporated under vacuum to yield 21.2 mg of the title compoundas a clear oil (81.9% crude yield). Thin layer chromatography analysisof the title compound showed a single spot with Rf=0.5 (silica; 90:10:2dichloromethane/methanol/acetic acid).

Example 36 Preparation of N-Boc--S--methyl-L-cysteine-L-proline-O-benzylester ##STR88##

The compound of Example 35 (14.64 g, 62.2 mmole) and L-proline-O-benzylester hydrochloride (15.04 g, 62.2 mmole) were suspended in 135 mL ofacetonitrile at 0° c., then BOP (27.51 g, 62.2 mmole) and NMM (18.9 g,186.6 mmole) were added. The ice bath was removed after 30 minutes andthe reaction was stirred for 18 hours at room temperature.

The reaction mixture solvent was evaporated at 25° c. under vacuum togive an oil which was dissolved in ethyl acetate (250 mL). This solutionwas successively washed with 1N hydrochloric acid (1×50 mL), saturatedsodium bicarbonate (1×50 mL) and brine (1×50 mL), dried with MgSO₄. Thesolution was evaporated under vacuum to give crude product.

The crude product was purified by column chromatography on silica gel,eluting with 1:1 hexane/ethyl acetate. 21.9 g (83.3% yield) of the titlecompound was isolated as an amorphous solid by evaporating the solventunder vacuum from the column fractions. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.53(silica, 3:2 ethyl acetate/hexane).

Example 37 Preparation of N-Boc-S-methyl-L-cysteinesulfone-L-proline-O-benzyl ester ##STR89##

The compound of Example 36 (16.13 g, 38.18 mmole) was dissolved in 200mL of glacial acetic acid and sodium perborate tetrahydrate (29.37 g,190.9 mmole) was added and the mixture heated to 55° C. After 2.5 hoursat this temperature, the reaction solution was diluted with 800 mL ofbrine, the aqueous layer was extracted with ethyl acetate (3×250 mL) andthe combined organic extracts were dried with MgSO₄. This solution wasfiltered, evaporated under vacuum to yield a residue that was repeatedlyazeotroped with toluene (200 mL) under vacuum to remove acetic acid. Theresidual slurry was suspended in ethyl acetate (200 mL), filtered andthe solvent evaporated under vacuum to yield 17.4 g (100% yield) of thetitle compound as a white solid. Thin layer chromatography analysis ofthe title compound showed a single spot with Rf=0.25 (3:2 ethylacetate/hexane).

Example 38 Preparation of N-(1-butanesulfonyl)-S-methyl-L-cysteinesulfone-L-proline-O-benzyl ester ##STR90##

The compound of Example 37 (2.0 g, 4.4 mmole) was dissolved in 12 mL of4N anhydrous hydrochloric acid/dioxane and was stirred for several hoursat room temperature until all starting material was consumed. Thehydrochloric acid/dioxane solution was then evaporated under vacuum toyield an oil. This oil was dissolved in acetonitrile and evaporatedunder vacuum. This was done three times.

The oil was suspended in 17 mL of acetonitrile, cooled to ice bathtemperature, then 1-butanesulfonyl chloride (0.69 g, 4.4 mmole) andpyridine (1.04 g, 13.2 mmole) were added. The reaction was taken fromthe ice bath after 30 minutes and stirred at room temperature for 18hours. The reaction mixture was evaporated under vacuum to give an oil.The oil was taken up in 200 mL ethyl acetate, and was washedsuccessively with 1N hydrochloric acid (1×50 mL) saturated sodiumbicarbonate 1×50 mL) and brine (1×50 mL). After drying with MgSO₄, thesolvent was evaporated under vacuum to give crude product.

This crude product was purified by column chromatography silica geleluting with 1:99; methanol/dichloromethane). Removal of the solventfrom the column fractions yielded 1.25 g (59.8% yield) of the titlecompound as a solid. Thin layer chromatography analysis of the titlecompound showed a single spot with Rf=0.59 (silica; 95:5dichloromethane/methanol).

Example 39 Preparation of N-(1-butanesulfonyl)-S-methyl-L-cysteinesulfone-L-proline ##STR91##

The compound of Example 38 (1.55 g, 2.45 mmole) was dissolved in THF (50mL), 0.5 g of 10% palladium on carbon was added and the mixture wasstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with 1N hydrochloric acid to pH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give 0.77 g (yield 82.9%)of the title compound as a foamy solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.3 (silica;90:10:2 dichloromethane/methanol/acetic acid).

Example 40 Preparation of N-(1-butanesulfonyl)-S-methyl-L-cysteinesulfone-L-proline-L-argininal ##STR92##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 39 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound. 0.1mL of thiocresol was additionally added to the anisole-HF cleavagemixture.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 524.2.

Example 41 Preparation of N-(2-propylpentanoyl)-S-methyl-L-cysteinesulfone-L-proline-O-benzyl ester ##STR93##

The compound of Example 37 (3.0 g, 4.66 mmole) was dissolved in 12 mL of4N anhydrous hydrochloric acid/dioxane. The solution was stirred forseveral hours at room temperature until all starting material wasconsumed. The hydrochloric acid/dioxane solution was evaporated undervacuum. This oil was dissolved in acetonitrile and evaporated undervacuum. This was done three times.

The oil was suspended in 25 mL of acetonitrile, cooled to ice bathtemperature, then 2-propylpentanoic acid (0.95 g, 6.6 mmole), BOP (2.92g, 6.6 mmole) and NMM (2.0 g, 19,8 mmole) were added. The reaction wastaken from the ice bath after 30 minutes and stirred at room temperaturefor 18 hours. The acetonitrile was evaporated under vacuum and the oilwas taken up in ethyl acetate (200 mL) and washed successively with 1Nhydrochloric acid (1×50 mL), saturated sodium bicarbonate (1×50 mL) andbrine (1×50 mL). After drying with MgSO₄, the ethyl acetate wasevaporated under vacuum to yield crude product.

The crude product was purified by column chromatography on silica gel,eluting with 1.5;98.5, methanol/dichloromethane). The solvent from thecolumn fractions was evaporated under vacuum to yield 2.0 g (63.0%yield) of the title compound as an oil. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.43(silica; 95:5:1 dichloromethane/methanol/acetic acid).

Example 42 Preparation of N-(2-propylpentanoyl)-S-methyl-L-cysteinesulfone-L-proline ##STR94##

The compound of Example 41 (1.75 g, 3.64 mmole) was dissolved in THF (50mL), 0.5 g of 10% palladium on carbon was added and the mixture wasstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with 1N hydrochloric acid to pH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give 0.90 g (63.1% yield)of the title compound as a foamy solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf 0.3 (silica,90:10:2 dichloromethane/methanol/acetic acid).

Example 43 Preparation of N-(2-propylpentanoyl)-S-methyl-L-cysteinesulfone-L-proline-L-argininal ##STR95##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 42 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound. 0.1mL of thiocresol was additionally added to the anisole-HF cleavagemixture.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 530.3.

Example 14 Preparation of N-(2-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-proline-O-benzyl ester ##STR96##

The compound of Example 37 (3.0 g, 6.6 mmole) was dissolved in 12 mL of4N anhydrous hydrochloric acid/dioxane. The solution was stirred forseveral hours at room temperature until all starting material wasconsumed. The hydrochloric acid/dioxane solution was evaporated undervacuum. The resulting oil was dissolved in acetonitrile and evaporatedunder vacuum. This was done three times.

The remaining oil was suspended in 20 mL of acetonitrile, cooled to icebath temperature, then 2-naphthalenesulfonyl chloride (0.1.49 g, 6.6mmole) and pyridine (1.57 g, 19.8 mmole) were added. The reaction wastaken from the ice bath after 30 minutes and stirred at room temperaturefor 18 hours. The reaction mixture was reduced in volume under vacuum toan oil. The oil was taken up in 200 mL ethyl acetate and washedsuccessively with 1N hydrochloric acid (1×50 mL), saturated sodiumbicarbonate 1×50 mL) and brine (1×50 mL). After drying with MgSO₄, theethyl acetate was evaporated under vacuum to give crude product.

The crude product was purified by column chromatography on silica gel,eluting with 0.8:99.2 methanol/dichloromethane to yield 2.2 g (60.6%yield) of the title compound as a solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.50(silica; 90:10:2 dichloromethane/methanol/acetic acid).

Example 45 Preparation of N-(2-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-proline ##STR97##

The compound of Example 44 (1.95 g, 3.58 mmole) was dissolved in THF (50mL), 0.5 g of 10% palladium on carbon was added and the mixture wasstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with 1N hydrochloric acid to pH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give 0.95 g of a solid(yield 58.3%). Thin layer chromatography of the isolated material wasperformed, Rf 0.3 (silica, 90:10:2 dichloromethane/methanol/aceticacid).

Example 46 Preparation of N-(2-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-proline-L-argininal ##STR98##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 45 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 594.2.

Example 47 Preparation of N-(1-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-proline-O-benzyl ester ##STR99##

The compound of Example 37 (3.0 g, 6.6 mmole) was dissolved in 12 mL of4N anhydrous hydrochloric acid/dioxane. The solution was stirred forseveral hours at room temperature until all starting material wasconsumed. The hydrochloric acid/dioxane solution was evaporated undervacuum to give an oil. The oil was dissolved in acetonitrile andevaporated under vacuum. This was done three times.

The remaining oil was suspended in 20 mL of acetonitrile, cooled to icebath temperature, then 1-naphthalenesulfonyl chloride (0.1.49 g, 6.6mmole) and pyridine (1.57 g, 19.8 mmole) were added. The reaction wastaken from the ice bath after 30 minutes and stirred at room temperaturefor 18 hours. The reaction mixture was reduced in volume under vacuum toan oil. The oil was taken up in 200 mL ethyl acetate and washedsuccessively with 1N hydrochloric acid (1×50 mL), saturated sodiumbicarbonate 1×50 mL) and brine (1×50 mL). After drying with MgSO₄, thesolvent was evaporated under vacuum to give crude product.

The crude product was purified by column chromatography on silica gel,eluting with 0.8:99.2 methanol/dichloromethane to yield 1.61 g (45%yield) of the title compound as a solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.50(silica; 90:10:2 dichloromethane/methanol/acetic acid).

Example 48 Preparation of N-(1-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-proline ##STR100##

The compound of Example 47 (1.6 g, 2.94 mmole) was dissolved in THF (50mL), 0.5 g of 10% palladium on carbon was added and the mixture wasstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with 1N hydrochloric acid to pH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give 1.07 g (80% yield)of the title compound. Thin layer chromatography analysis of the titlecompound showed a single spot with Rf=0.3 (silica; 90:10:2dichloromethane/methanol/acetic acid).

Example 49 Preparation of N-(1-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-proline-L-argininal ##STR101##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 48[N-(1-naphthalene-sulfonyl)-S-methyl-L-cysteine sulfone-L-proline] wasattached to the resin of Example 7. After cleavage of the title compoundas a protected semicarbazone from the resin, the semicarbazone washydrolyzed to give the title compound. 0.1 mL of thiocresol wasadditionally added to the anisole-HF cleavage mixture.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 594.2.

Example 50 Preparation of S-(t-butyl acetate)-L-cysteine ##STR102##

A 360 mL aqueous solution of commercially available (Aldrich) L-cysteinehydrochloride monohydrate (60.0 g, 341.7 mmole) and sodium hydroxide(27.33 g, 683.4 mmole), at room temperature, was treated with a solutionof t-butyl bromoacetate (72.3 g, 370.6 mmole) in 130 mL of dioxane over30 minutes. The reaction was stirred for 18 hours, during which time athick precipitate formed. The solid was filtered off, washed withdiethyl ether (100 mL) and dried under high vacuum at 40° C. to give82.5 g (103.8% crude yield includes occluded inorganic salt) of thetitle compound.

Example 51 Preparation of N-Boc-S-(t-butyl acetate)-L-cysteine##STR103##

The compound of Example 50 (82.5 g, 341.7 mmole) and sodium bicarbonate(33.96 g, 404 mmole) were suspended in 600 mL of deionized water. Asolution of di-t-butyl dicarbonate (80.88 g, 370 mmole) in 350 mL ofdioxane was added and the slurry was stirred for 18 hours.

The slurry was extracted with diethyl ether (2×100 mL). The slurry waslayered with ethyl acetate (200 mL) and acidified with 1N hydrochloricacid to pH 2 (pH papers). The resulting organic layer was saved and theremaining aqueous layer was further extracted with ethyl acetate (2×200mL). The organic extracts were combined, washed with brine, dried withMgSO₄ and the solvent evaporated under vacuum to yield 84.3 g (74.6%yield) of the title compound as a clear oil. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.55,(silica; 90:10:2 dichloromethane/methanol/acetic acid).

Example 52 Preparation of N-Boc-S-(t-butylacetate)-L-cysteine-L-proline-O-benzyl ester ##STR104##

The compound of Example 51 (31.89 g, 95.06 mmole) and L-proline-O-benzylester hydrochloride (22.98 g, 95.06 mmole) were suspended in 140 mL ofacetonitrile and 120 mL of DMF at 0° c., then BOP (42.0 g, 95.06 mmole)and NMM (28.84 g, 285.18 mmole) were added. The ice bath was removedafter 30 minutes and the reaction was stirred for 18 hours at roomtemperature. The reaction mixture was reduced in volume under vacuum at25° C. to give an oil. The oil was dissolved in ethyl acetate (250 mL),then successively washed with 1N hydrochloric acid (1×50 mL), saturatedsodium bicarbonate (1×50 mL) and brine (1×50 mL). The organic layer wasdried with MgSO₄ and evaporated under vacuum to give crude product.

The crude product was purified by column chromatography on silica gel,eluting with 55:45 hexane/ethyl acetate to yield 27.91 g (57.9% yield)of the title compound as an oil. Thin layer chromatography analysis ofthe title compound showed a single spot with Rf=0.65 (silica, 3:2 ethylacetate/hexane).

Example 53 Preparation of N-Boc-S-(t butyl acetate)-L-cysteinesulfone-L-proline-O-benzyl ester ##STR105##

The compound of Example 52 (27.9 g, 55.07 mmole) was dissolved in 300 mLof glacial acetic acid, sodium perborate tetrahydrate (42.36 g, 275.35mmole) was added and the mixture was heated to 55° C. After 2.5 hours atthis temperature, the reaction mixture was diluted with 1 liter ofbrine, the aqueous layer was extracted with ethyl acetate (4×250 mL) andthe combined organic extracts were dried with MgSO₄. This solution wasfiltered and evaporated under vacuum, then repeatedly azeotroped withtoluene (200 mL) under vacuum to remove acetic acid. The residual slurrywas dissolved in ethyl acetate (200 mL), filtered and the filtrateevaporated to yield 29.7 g (100% yield) of the title compound as a whitesolid. Thin layer chromatography analysis of the title compound showed asingle spot with Rf=0.60 (silica, 3:2 ethyl acetate/hexane).

Example 54 Preparation of N-benzylsulfonyl-S(t-butyl acetate)-L-cysteinesulfone-L-proline-O-benzyl ester ##STR106##

A solution of the compound of Example 53 (5.0 g, 9.28 mmole) in 105 mLof sieve-dried ethyl acetate was prepared. To this, 26 mL of 5.7Nanhydrous hydrochloric acid/ethyl acetate (that had been generated insitu from acetyl chloride and methanol) was added. This mixture wasstirred for several hours at room temperature until all startingmaterial was consumed. The mixture was evaporated under vacuum and theresulting oil was dissolved in acetonitrile and then evaporated undervacuum. This was done three times.

The remaining oil was suspended in 35 mL of acetonitrile, cooled to icebath temperature, then benzylsulfonyl chloride (2.12 g, 11.14 mmole) andpyridine (2.93 g, 37.12 mmole) were added. The reaction was removed fromthe ice bath after 30 minutes and allowed stirred at room temperaturefor 18 hours. The reaction mixture was reduced in volume under vacuum toan oil. The oil was taken up in 200 mL ethyl acetate and washedsuccessively with IN hydrochloric acid (1×50 mL), saturated sodiumbicarbonate (1×50 mL) and brine (1×50 mL). After drying with MgSO₄, theorganic layer was evaporated under vacuum to give crude product.

The crude product was purified by column chromatography on silica gel,eluting with 3:2 hexane/ethyl acetate to yield 2.85 g (51.8% yield) ofthe title compound as a solid. Thin layer chromatography analysis of thetitle compound showed a single spot with Rf=0.30 (silica, 3:2 ethylacetate/hexane).

Example 55 Preparation of N-benzylsulfonyl-S-(t-butylacetate)-L-cysteine sulfone-L-proline ##STR107##

The compound of Example 54 (3.85 g, 4.81 mmole) was dissolved in THF (50mL), 0.5 g of 10% palladium on carbon was added and the mixture wasstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with 1N hydrochloric acid to pH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give 2.1 g (yield 86.9%)of the title compound as a foamy solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.35(silica, 90:10:2 dichloromethane/methanol/acetic acid).

Example 56 Preparation of N-benzylsulfonyl-S-(carboxymethyl)-L-cysteinesulfone-L-proline-L-argininal ##STR108##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 55 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 602.

Example 57 Preparation of N-(2-propylpentanoyl-S-(t-butylacetate)-L-cysteine sulfone-L-proline-O-benzyl ester ##STR109##

A solution of the compound of Example 53 (5.0 g, 9.28 mmole) in 105 mLof sieve-dried ethyl acetate was prepared. To this, 26 mL of 5.7Nanhydrous hydrochloric acid/ethyl acetate (that had been generated insitu from acetyl chloride and methanol) was added. This mixture wasstirred for several hours at room temperature until all startingmaterial was consumed. The mixture was evaporated under vacuum and theresulting oil was dissolved in acetonitrile and then evaporated undervacuum. This was done three times.

The remaining oil was suspended in 35 mL of acetonitrile, cooled to icebath temperature, then 2-propylpentanoic acid (1.60 g, 11.4 mmole), BOP(4.10 g, 9.28 mmole) and NMM (3.75 g, 37.1 mmole) were added. Thereaction was removed from the ice bath after 30 minutes and allowedstirred at room temperature for 18 hours. The reaction mixture wasreduced in volume under vacuum to an oil. The oil was taken up in 200 mLethyl acetate and washed successively with 1N hydrochloric acid (1×50mL), saturated sodium bicarbonate (1×50 mL) and brine (1×50 mL). Afterdrying with MgSO₄, the organic layer was evaporated under vacuum to givecrude product.

The crude product was purified by column chromatography on silica gel,eluting with 3:2 hexane/ethyl acetate to yield 1.81 g (34.5% yield) ofthe title compound as a solid. Thin layer chromatography analysis of thetitle compound showed a single spot with Rf=0.50 (silica, 3:2 ethylacetate/hexane).

Example 58 Preparation of N-(2-propylpentanoyl)-S-(t-butylacetate)-L-cysteine sulfone-L-proline ##STR110##

The compound of Example 57 (1.81 g, 3.2 mmole) was dissolved in THF (50mL), 0.5 g of 10% palladium on carbon was added and the mixture wasstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with 1N hydrochloric acid to pH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give 1.30 g (yield 85.6%)of the title compound as a foamy solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.35(silica, 90:10:2 dichloromethane/methanol/acetic acid).

Example 59 Preparation ofN-(2-propylpentanoyl)-S-(carboxymethyl)-L-cysteinesulfone-L-proline-L-argininal ##STR111##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 58 was attached to the resin of Example 7. Aftercleavage of the title compound as a protected semicarbazone from theresin, the semicarbazone was hydrolyzed to give the title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 574.

Example 60 Preparation of S-(methyl acetate)-L-cysteine ##STR112##

A 70 mL aqueous solution of commercially available (Aldrich) L-cysteinehydrochloride monohydrate (25.0 g, 140 mmole) and sodium hydroxide (11.2g, 280 mmole) at room temperature was treated with a solution of methylbromoacetate (25.7 g, 168 mmole) in 70 mL of dioxane over 30 minutes.After stirring for 18 hours, the solution was extracted with diethylether (2×50 mL) and evaporated under vacuum at 35° C. to give the titlecompound as a solid. This was used in the next reaction without furtherpurification.

Example 61 Preparation of N-Boc-S-(methyl acetate)-L-cysteine ##STR113##

The compound of Example 60 (27.0 g, 140 mmole) was suspended in 155 mLof saturated sodium bicarbonate (approximately 1.1M). A solution ofdi-t-butyl dicarbonate (35.15 g, 161 mmole) in 75 mL of THF was addedand the mixture was vigorously stirred for 18 hours at room temperature.

The reaction mixture was extracted with diethyl ether (100 mL) and theorganic phase decanted off. The aqueous phase was then layered withethyl acetate (200 mL) and was acidified to pH 2 (pH papers) with 1Nhydrochloric acid. After the layers were separated, the organic layerwas saved and the aqueous layer was extracted with ethyl acetate (3×100mL). The organic extracts were combined and washed with brine, driedwith MgSO₄ and the solvent was evaporated under vacuum to yield 20.66 g(50% crude yield) of the title compound as a clear oil. Thin layerchromatography analysis of the title compound showed a single spot withRf=0.5 (silica, 90:10:2 dichloromethane/methanol/acetic acid).

Example 62 Preparation of N-Boc-S-(methylacetate)-L-cysteine-L-proline-O-benzyl ester ##STR114##

The compound of Example 61 (20.69 g, 70.5 mmole) and L-proline-O-benzylester hydrochloride (16.92 g, 70.5 mmole) were suspended in 140 mL ofacetonitrile at 0° C., then BOP (30.96 g, 70.5 mmole) and NMM (21.2 g,211 mmole) were added. The ice bath was removed after 30 minutes and thereaction was stirred for 18 hours at room temperature. The reactionmixture was reduced in volume at 25° C. under vacuum to give an oil. Theoil was dissolved in ethyl acetate (250 mL), then successively washedwith 1N hydrochloric acid (1×50 mL), saturated sodium bicarbonate (1×50mL) and brine (1×50 mL). The organic layer was dried with MgSO₄ andevaporated under vacuum to give crude product.

This crude product was purified by column chromatography on silica gel,eluting with 99:1 dichloromethane/methanol to yield 15.6 g (46.0% yield)of the title compound as an oil. Thin layer chromatography analysis ofthe title compound showed a single spot with Rf=0.50 (silica, 95:5dichloromethane/methanol).

Example 63 Preparation of N-Boc-S-(methyl acetate-L-cysteinesulfone-L-proline-O-benzyl ester ##STR115##

The compound of Example 62 (15.16 g, 31.55 mmole) was dissolved in 165mL of glacial acetic acid, sodium perborate tetrahydrate (24.27 g,157.75 mmole) was added and the mixture was heated to 55° C. After 2.5hours at this temperature, the reaction solution was diluted with 700 mLof brine, the aqueous layer was extracted with ethyl acetate (3×250 mL)and the combined organic phases were dried with MgSO₄. This solution wasfiltered and evaporated under vacuum, then repeatedly azeotroped withtoluene (200 mL) under vacuum to remove acetic acid. The residual slurrywas dissolved in ethyl acetate (200 mL), filtered and the filtrateevaporated under vacuum to yield 15.95 g (98.6% yield) of the titlecompound as an oil. Thin layer chromatography analysis of the titlecompound showed a single spot with Rf=0.30 (silica, 3:2 ethylacetate/hexane).

Example 64 Preparation of N-benzylsulfonyl-S-(methyl acetate)-L-cysteinesulfone-L-proline-O-benzyl ester ##STR116##

The compound of Example 63 (5.66 g, 10.9 mmole) was dissolved in 18 mLof 4N anhydrous hydrochloric acid/dioxane. The solution was stirred forseveral hours at room temperature until all starting material wasconsumed. The hydrochloric acid/dioxane solution was evaporated undervacuum to give an oil. The oil was dissolved in acetonitrile andevaporated under vacuum. This was done three times.

The remaining oil was suspended in 25 mL of acetonitrile, cooled to icebath temperature, then benzylsulfonyl chloride (2.5 g, 13.1 mmole) andpyridine (2.6 g, 32.8 mmole) were added. The reaction was taken from theice bath after 30 minutes and stirred at room temperature for 18 hours.The acetonitrile was evaporated under vacuum and the resulting oil wastaken up in 200 mL ethyl acetate and washed successively with 1Nhydrochloric acid (1×50 mL), saturated sodium bicarbonate 1×50 mL) andbrine (1×50 mL). After drying with MgSO₄, the solvent was evaporatedunder vacuum to give crude product.

The crude product was purified by column chromatography on silica gel,eluting with 1:1 hexane/ethyl acetate to yield 2.9 g (46.9% yield) ofthe title compound as solid. Thin layer chromatography analysis of thetitle compound showed a single spot with Rf=0.27 (silica; 3:2 ethylacetate/hexane).

Example 65 Preparation of N-benzylsulfonyl-S-(methyl acetate)-L-cysteinesulfone-L-proline ##STR117##

The compound of Example 64 (2.9 g, 5.12 mmole) was dissolved in THF (50mL), 0.5 g of 10% palladium on carbon was added and the mixture wasstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with 1N hydrochloric acid to pH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give 2.17 g (yield 88.9%)of the title compound as a foamy solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.40(silica, 90:10:2 dichloromethane/methanol/acetic acid).

Example 66 Preparation of N-benzylsulfonyl-S-(methyl acetate)-L-cysteinesulfone-L-proline-L-argininal ##STR118##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 65 is attached to the resin of Example 7. Thetitle compound as a protected semicarbazone is then be cleaved from theresin and the semicarbazone is hydrolyzed to give the title compound.

Example 67 Preparation of N-(2-naphthylenesulfonyl)-S-(methylacetate)-L-cysteine sulfone-L-proline-O-benzyl ester ##STR119##

The compound of Example 63 (4.8 g, 9.37 mmole) was dissolved in 18 mL of4N anhydrous hydrochloric acid/dioxane. The solution was stirred forseveral hours at room temperature until all starting material wasconsumed. The hydrochloric acid/dioxane solution was evaporated undervacuum to give an oil. The oil was dissolved in acetonitrile andevaporated under vacuum. This was done three times.

The remaining oil was suspended in 35 mL of acetonitrile, cooled to icebath temperature, then 2-naphthylenesulfonyl chloride (2.55 g, 11.2mmole) and pyridine (2.2 g, 28.1 mmole) were added. The reaction wastaken from the ice bath after 30 minutes and stirred at room temperaturefor 18 hours. The acetonitrile was evaporated under vacuum and theresulting oil was taken up in 200 mL ethyl acetate and washedsuccessively with 1N hydrochloric acid (1×50 mL), saturated sodiumbicarbonate 1×50 mL) and brine (1×50 mL). After drying with MgSO₄, thesolvent was evaporated under vacuum to give crude product.

The crude product was purified by column chromatography on silica gel,eluting with 99.25:0.75 dichloromethane/methanol to yield 3.9 g (69.0%yield) of the title compound as solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.35(silica; 3:2 ethyl acetate/hexane).

Example 68 Preparation of N-(2-naphthylenesulfonyl)-S-(methylacetate)-L-cysteine sulfone-L-proline ##STR120##

The compound of Example 67 (3.90 g, 6.47 mmole) was dissolved in THF (50mL), 0.5 g of 10% palladium on carbon was added and the mixture wasstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst was filtered off the reaction mixture, the solventwas removed under vacuum and the resulting oil was taken up in asolution of saturated sodium bicarbonate. This solution was thenextracted with ethyl acetate (1×150 mL) and the organic layer wasdecanted off. The remaining aqueous layer was layered with 100 mL ofethyl acetate and acidified with 1N hydrochloric acid to PH 2 (pHpapers). After the phases separated, the organic layer was saved and theaqueous layer was then further extracted with ethyl acetate (3×100 mL).

The organic extracts were combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give 2.84 g (yield 85.8%)of the title compound as a foamy solid. Thin layer chromatographyanalysis of the title compound showed a single spot with Rf=0.40(silica, 90:10:2 dichloromethane/methanol/acetic acid).

Example 69 Preparation of N-(2-naphthylenesulfonyl)-S-(methylacetate)-L-cysteine sulfone-L-proline-L-argininal ##STR121##

The title compound was prepared in the same manner as described inExample 8.

The compound of Example 68 [N-(2-naphthylene-sulfonyl)-S-(methylacetate)-L-cysteine sulfone-L-proline] was attached to the resin ofExample 7. After cleavage of the title compound as a protectedsemicarbazone from the resin, the semicarbazone was hydrolyzed to givethe title compound.

Fast atom bombardment mass spectrometry confirmed the theoreticalmolecular weight of 652.

Example 70 Preparation of S-(cyanomethyl)-L-cysteine ##STR122##

A 360 mL aqueous solution of commercially available (Aldrich) L-cysteinehydrochloride monohydrate (60.0 g, 341.7 mmole) and sodium hydroxide(27.33 g, 683.4 mmole), at room temperature, is treated with a solutionof bromoacetonitrile (44.5 g, 370.6 mmole) in 130 mL of dioxane over 30minutes. The reaction is stirred for 18 hours, during which time a thickprecipitate will form. The solid is filtered off, washed with diethylether (100 mL) and dried under high vacuum at 40° C. to give the titlecompound.

Example 71 Preparation of N-Boc-S-(cyanomethyl)-L-cysteine ##STR123##

The compound of Example 70 (54.7 g, 341.7 mmole) and sodium bicarbonate(33.96 g, 404 mmole) are suspended in 600 mL of deionized water. Asolution of di-t-butyl dicarbonate (80.88 g, 370 mmole) in 350 mL ofdioxane is added and the slurry is stirred for 18 hours.

The slurry is extracted with diethyl ether (2×100 mL). The slurry islayered with ethyl acetate (200 mL) and acidified with 1N hydrochloricacid to pH 2 (pH papers). The resulting organic layer is saved and theremaining aqueous layer is further extracted with ethyl acetate (2×200mL). The organic extracts are combined, washed with brine, dried withMgSO₄ and the solvent evaporated under vacuum to give the titlecompound.

Example 72 Preparation ofN-Boc-S-(cyanomethyl)-L-cysteine-L-proline-O-benzyl ester ##STR124##

The compound of Example 71 (24.72, 95.06 mmole) and L-proline-O-benzylester hydrochloride (22.98 g, 95.06 mmole) are suspended in 140 mL ofacetonitrile and 120 mL of DMF at 0° C., then BOP (42.0 g, 95.06 mmole)and NMM (28.84 g, 285.18 mmole) are added. The ice bath is removed after30 minutes and the reaction is stirred for 18 hours at room temperature.The reaction mixture is reduced in volume under vacuum at 25° C. to givea residue. The residue is taken up in 200 mL ethyl acetate, and iswashed successively with 1N hydrochloric acid (1×50 mL) saturated sodiumbicarbonate 1×50 mL) and brine (1×50 mL). After drying with MgSO₄, thesolvent is evaporated under vacuum to give the title compound.

Example 73 Preparation of N-Boc-S-(cyanomethyl)-L-cysteinesulfone-L-proline-O-benzyl ester ##STR125##

The compound of Example 72 (23.96 g, 55.07 mmole) is dissolved in 300 mLof glacial acetic acid, sodium perborate tetrahydrate (42.36 g, 275.35mmole) is added and the mixture is heated to 55° C. After 2.5 hours atthis temperature, the reaction mixture is diluted with 1 liter of brine,the aqueous layer is extracted with ethyl acetate (4×250 mL) and thecombined organic extracts are dried with MgSO₄. This solution isfiltered and evaporated under vacuum, then repeatedly azeotroped withtoluene (200 mL) under vacuum to remove acetic acid. The residue isdissolved in ethyl acetate (200 mL), filtered and the filtrate isevaporated under vacuum to give the title compound.

Example 74 Preparation of N-benzylsulfonyl-S-(cyanomethyl)-L-cysteinesulfone-L-proline-O-benzyl ester ##STR126##

A solution of the compound of Example 73 (4.3 g, 9.28 mmole) in 105 mLof sieve-dried ethyl acetate is prepared. To this, 26 mL of 5.7Nanhydrous hydrochloric acid/ethyl acetate (that is generated in situfrom acetyl chloride and methanol) is added. This mixture is stirred forseveral hours at room temperature until all starting material isconsumed. The mixture is evaporated under vacuum and the residue isdissolved in acetonitrile and then evaporated under vacuum. This wasdone three times to give a further residue.

The residue is suspended in 35 mL of acetonitrile, cooled to ice bathtemperature, then benzylsulfonyl chloride (2.12 g, 11.14 mmole) andpyridine (2.93 g, 37.12 mmole) are added. The reaction is removed fromthe ice bath after 30 minutes and allowed to stir at room temperaturefor 18 hours. The reaction mixture is reduced in volume under vacuum toa residue which is taken up in 200 mL ethyl acetate and washedsuccessively with 1N hydrochloric acid (1×50 mL), saturated sodiumbicarbonate (1×50 mL) and brine (1×50 mL). After drying with MgSO₄, thesolvent is evaporated under vacuum to give the title compound.

Example 75 Preparation of N-benzylsulfonyl-S-(tetrazol-5-yl)-L-cysteinemethyl sulfone-L-proline-O-benzyl ester ##STR127##

To the compound of Example 74 (5.3 g, 10.0 mmole) which is dissolved in20 mL of THF is added tributyltin azide (4.71 g, 15.0 mmole). Thereaction mixture is refluxed for three days. The reaction mixture isallowed to cool and the volatiles are removed under vacuum. The residueis dissolved in 50 mL of saturated sodium bicarbonate and is washed withethyl acetate (3×25 mL). The aqueous phase is then acidified to pH 3with 1N hydrochloric acid, then is extracted with ethyl acetate (3×75mL). The combined organic extracts are dried over MgSO₄ and the solventis removed under vacuum to give the title compound.

Example 76 Preparation of N-benzylsulfonyl-S-(tetrazol-5-yl)-L-cysteinemethyl sulfone-L-proline ##STR128##

The compound of Example 75 (2.77 g, 4.81 mmole) is dissolved in THF (50mL), 0.5 g of 10% palladium on carbon is added and the mixture isstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered off the reaction mixture, the solvent isremoved under vacuum and the resulting residue is taken up in a solutionof saturated sodium bicarbonate. This solution is then extracted withethyl acetate (1×150 mL) and the organic layer is decanted off. Theremaining aqueous layer is layered with 100 mL of ethyl acetate andacidified with 1N hydrochloric acid to pH 2 (pH papers). After thephases separate, the organic layer is saved and the aqueous layer isthen further extracted with ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 77 Preparation of N-benzylsulfonyl-S-(tetrazol-5-yl)-L-cysteinemethyl sulfone-L-proline-L-argininal ##STR129##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 76 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 78 Preparation of N-Boc-L-2-azetidinecarboxylic acid ##STR130##

L-2-azetidinecarboxylic acid (34.5 g, 341.7 mmole) and sodiumbicarbonate (33.96 g, 404 mmole) are suspended in 600 mL of deionizedwater. A solution of di-t-butyl dicarbonate (80.88 g, 370 mmole) in 350mL of dioxane is added and the slurry is stirred for 18 hours.

The slurry is extracted with diethyl ether (2×100 mL). The slurry islayered with ethyl acetate (200 mL) and acidified with 1N hydrochloricacid to pH 2 (pH papers). The resulting organic layer is saved and theremaining aqueous layer is further extracted with ethyl acetate (2×200mL). The organic extracts are combined, washed with brine, dried withMgSO₄ and the solvent evaporated under vacuum to give the titlecompound.

Example 79 Preparation of N-Boc-L-2-azetidinecarboxylic acid-O-benzylester ##STR131##

To a solution of compound of Example 78 (178 mmole) in dry THF (500 mL)which is chilled to 0° C., carbonyl diimidazole (34.6 g, 214 mmole) isadded in small portions. After 30 minutes, the mixture is warmed to roomtemperature for 2 hours until all of the CO₂ evolution ceases. Afterthis time, benzyl alcohol (27.6 mL, 267 mmole) is added and the reactionis stirred for 12 hours.

The reaction mixture is then reduced in volume under vacuum and theresidue is diluted with ethyl acetate (500 mL). The organic phase arewashed with saturated bicarbonate (1×100 mL), brine (100 mL), saturatedaqueous citric acid (1×100 mL), dried over MgSO₄, filtered and thesolvent is removed under vacuum to give the title compound.

Example 80 Preparation of L-2-azetidinecarboxylic acid-O-benzyl esterhydrochloride salt ##STR132##

The compound of Example 79 (50.0 g) is dissolved in 200 mL of a 4Msolution of HCl in dioxine. The reaction mixture is stirred 2 hours. Thesolution is reduced in volume under vacuum. The resulting solid iswashed with diethyl ether to give the title compound.

Example 81 Preparation of L-methionine sulfone-L-2-azetidinecarboxylicacid-O-benzyl ester hydrochloride salt ##STR133## A. Procedure 1:

N-Boc-L-methionine sulfone-L-2-azetidinecarboxylic acid-O-benzyl esteris prepared by adding to a solution of N-Boc-L-methioninesulfone (14.0g, 50.0 mmole) in dichloromethane (150 mL) at 0° C., HOBt (10.1 g, 75mmole) followed by DCC (11.33 g, 55.0 mmole). The mixture is stirred for10 minutes, and then the compound of Example 80 (50.0 mmole) is addedfollowed by NMM (100 mmole, 10.9 mL). The resulting mixture is stirredin an ice bath and is allowed to come to room temperature over 12 hours.The mixture is filtered to remove dicyclohexylurea and ethyl acetate(300 mL) is added. The organic phase is then added to a separatoryfunnel and is washed with saturated aqueous sodium bicarbonate, brineand then 1M aqueous HCl. The organic phase is dried over magnesiumsulfate and then is filtered. The organic phase is reduced on a rotaryevaporator under vacuum and then on a high vacuum line to remove tracesof solvent to give N-Boc-L-methionine sulfone-L-2-azetidinecarboxylicacid-O-benzyl ester.

To a solution of N-Boc-L-methionine sulfone-L-2-azetidinecarboxylicacid-O-benzyl ester (50 mmole) in dry dioxane (300 mL) is added 100 mLof a 4M HCl/dioxane solution. The mixture is then stirred at roomtemperature for 1 hour until the starting material disappears. Diethylether is added to the mixture to precipitate the title compound. Themixture is filtered off on a Buchner funnel and dried under high vacuumto give the title compound.

B. Procedure 2:

Alternatively, the title compound is synthesized by the followingmethod.

To a solution of N-Boc-L-methionine sulfone (5 g, 20 mmole) in 80 mL ofdry DMF is added compound of Example 80 (20 mmole) followed by BOP (8.9g, 20 mmole) and NMM (5.5 mL, 20 mmole). The mixture is stirred for 16hours at room temperature. The reaction mixture is dissolved in 600 mLof ethyl acetate and is washed with 200 mL each of water, 1M aqueous HClwater, saturated aqueous sodium bicarbonate and brine. The organic phaseis dried over magnesium sulfate, filtered and the solvent is removedunder vacuum to give a residue.

To the solution of the residue in 20 mL dichloromethane, 100 mL of a 4Msolution of HCl in dioxane is added. After stirring for 16 hours, thesolvent is removed under vacuum. The resulting residue is precipitatedusing diethyl ether, filtered and dried under vacuum to give the titlecompound.

Example 82 Preparation ofN-(1-butanesulfonyl)-L-methioninesulfone-L-2-azetidinecarboxylic acid##STR134##

The compound of Example 81 (12.4 mmole) is reacted with 2.07 mL (16mmole) of 1-butanesulfonylchloride and 5.0 mL (36 mmole) oftriethylamine in dichloromethane from 0° C. to room temperature. Thereaction mixture is poured into saturated aqueous bicarbonate andextracted with ethyl acetate (2×100 mL). The organic phase is washedwith brine and 1M aqueous HCl. The organic phase is separated and driedover magnesium sulfate, filtered and reduced under vacuum to give aresidue.

The residue is mixed with 2M potassium hydroxide (20 mL) and 100 mLmethanol at room temperature for two hours. The methanol is reducedunder vacuum and the aqueous solution is then washed with ether (2×50mL) and then neutralized with 1M HCl to a pH of 1. The aqueous solutionis extracted with ethyl acetate (2×100 mL), dried over MgSO₄, filteredand reduced under vacuum to give the title compound.

Example 83 Preparation ofN-(1-butanesulfonyl)-L-methioninesulfone-L-2-azetidinecarboxylicacid-L-argininal ##STR135##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 82 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 84 Preparation of N-(2-naphthylenesulfonyl)-L-methioninesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR136##

The compound of Example 81 (6.9 mmole) is added to 69 mL ofacetonitrile. To this mixture, 2.34 g (10.3 mmole, 1.5 eq.) of2-naphthylenesulfonylchloride and 4.20 g (4.12 mL, 34.4 mmole, 5 eq.) ofpyridine are added and the mixture is stirred for 10 hours. The mixtureis then concentrated under vacuum, diluted with ethyl acetate (500 mL)and washed with 1M HCl water, aqueous sodium bicarbonate, and brine. Theorganic phase is dried over MgSO₄ and concentrated under vacuum to givea residue. The residue is then filtered down a plug of silica, elutingwith dichloromethane (100 mL), then 10% methanol/dichloromethane (200mL) to give the title compound.

Example 85 Preparation of N-(2-naphthylenesulfonyl)-L-methioninesulfone-L-2-azetidinecarboxylic acid ##STR137##

The compound of Example 84 (7.1 mmole) is dissolved in 250 mL ofmethanol with a trace of THF. To this solution, 2 g of 10% palladium oncarbon is added under a nitrogen blanket. The mixture is then stirredunder hydrogen at one atmosphere of pressure for 10 hours. The mixtureis filtered through a nylon filter and concentrated under vacuum to givethe title compound.

Example 86 Preparation of N-(2-naphthylenesulfonyl)-L-methioninesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR138##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 85 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 87 Preparation of N-benzylsulfonyl-L-methioninesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR139##

To a solution of the compound of Example 81 (20.0 mmole) in dryacetonitrile (100 mL) cooled to O° C., alpha-toluenesulfonylchloride(20.0 mmole, 3.8 g) is added all at once followed by pyridine (50.0mmole, 4.2 mL). The mixture is then stirred in the ice bath for 12 hoursduring which time the mixture is allowed to warm to room temperature.

The reaction mixture is reduced to a residue under vacuum. The residueis taken up ethyl acetate (300 mL) and washed with saturated aqueoussodium bicarbonate, brine, 1M aqueous HCl (100 mL), dried over MgSO₄,filtered and evaporated under vacuum to give the title compound. Thesolid is filtered through a plug of silica gel (50 g) using ethylacetate as eluent before hydrogenation to eliminate possible sulfurrelated impurities.

Example 88 Preparation of N-benzylsulfonyl-L-methioninesulfone-L-2-azetidinecarboxylic acid ##STR140##

To a solution of the compound of Example 87 (20 mmole) in methanol (300mL), 1.0 g of 10% palladium on carbon is added. The mixture is thenhydrogenated at 1 atmosphere of hydrogen gas at room temperature for 12hours with stirring. The mixture is filtered and reduced under vacuum togive the title compound.

Example 89 Preparation of N-benzylsulfonyl-L-methioninesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR141##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 88 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 90 Preparation of N-(2-propylpentanoyl)-L-methioninesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR142##

The compound of Example 81 (4.66 mmole) is dissolved in 12 mL of 4Nanhydrous hydrochloric acid/dioxane. The solution is stirred for severalhours at room temperature until all starting material is consumed. Thehydrochloric acid/dioxane solution is evaporated under vacuum to give aresidue. The residue is dissolved in acetonitrile and evaporated undervacuum. This is done three times to give a residue.

The residue is suspended in 25 mL of acetonitrile, cooled to ice bathtemperature, then 2-propylpentanoic acid (0.95 g, 6.6 mmole), BOP (2.92g, 6.6 mmole) and NMM (2.0 g, 19,8 mmole) are added. The reaction istaken from the ice bath after 30 minutes and stirred at room temperaturefor 18 hours. The acetonitrile is evaporated under vacuum and theresidue is taken up in ethyl acetate (200 mL) and washed successivelywith 1N hydrochloric acid (1×50 mL), saturated sodium bicarbonate (1×50mL) and brine (1×50 mL). After drying with MgSO₄, the ethyl acetate isevaporated under vacuum to yield crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 91 Preparation of N-(2-propylpentanoyl)-L-methioninesulfone-L-2-azetidinecarboxylic acid ##STR143##

The compound of Example 90 (3.64 mmole) is dissolved in THF (50 mL), 0.5g of 10% palladium on carbon is added and the mixture is stirred underhydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered from the reaction mixture, the solvent isremoved under vacuum and the residue is taken up in a solution ofsaturated sodium bicarbonate. This solution is then extracted with ethylacetate (1×150 mL) and the organic layer is decanted off. The remainingaqueous layer is layered with 100 mL of ethyl acetate and acidified with1N hydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 92 Preparation of N-(2-propylpentanoyl)-L-methioninesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR144##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 91 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound. 0.1 mL ofthiocresol is additionally added to the anisole-HF cleavage mixture.

Example 93 Preparation ofN-Boc-S-methyl-L-cysteine-L-2-azetidinecarboxylic acid-O-benzyl ester##STR145##

The compound of Example 35 [N-Boc-S-methyl-L-cysteine] (14.64 g, 62.2mmole) and the compound of Example 80 [L-2-azetidinecarboxylicacid-O-benzyl ester] (62.2 mmole) are suspended in 135 mL ofacetonitrile at 0° C., then BOP (27.51 g, 62.2 mmole) and NMM (18.9 g,186.6 mmole) are added. The ice bath is removed after 30 minutes and thereaction is stirred for 18 hours at room temperature.

The solvent from reaction mixture is evaporated at 25° C. under vacuumto give a residue which is dissolved in ethyl acetate (250 mL). Thissolution is successively washed with 1N hydrochloric acid (1×50 mL),saturated sodium bicarbonate (1×50 mL) and brine (1×50 mL), and then isdried with MgSO₄. The solvent is evaporated under vacuum to give crudeproduct.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 94 Preparation of N-Boc-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR146##

The compound of Example 93 (38.2 mmole) is dissolved in 200 mL ofglacial acetic acid and sodium perborate tetrahydrate (29.37 g, 190.9mmole) is added and the mixture is heated to 55° C. After 2.5 hours, atthis temperature, the reaction solution is diluted with 800 mL of brine,the aqueous layer is extracted with ethyl acetate (3×250 mL) and thecombined organic extracts are dried with MgSO₄. This solution isfiltered, evaporated under vacuum to yield a residue that is repeatedlyazeotroped with toluene (200 mL) under vacuum to remove acetic acid. Theresidual slurry is suspended in ethyl acetate (200 mL), filtered and thesolvent is evaporated under vacuum to give the title compound.

Example 95 Preparation of N-(1-butanesulfonyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR147##

The compound of Example 94 (4.4 mmole) is dissolved in 12 mL of 4Nanhydrous hydrochloric acid/dioxane and is stirred for several hours atroom temperature until all starting material is consumed. Thehydrochloric acid/dioxane solution is then evaporated under vacuum toyield a residue. This residue is dissolved in acetonitrile andevaporated under vacuum. This is done three times to give a furtherresidue.

The residue is suspended in 17 mL of acetonitrile, cooled to ice bathtemperature, then 1-butanesulfonyl chloride (0.69 g, 4.4 mmole) andpyridine (1.04 g, 13.2 mmole) are added. The reaction is taken from theice bath after 30 minutes and is stirred at room temperature for 18hours. The reaction mixture is evaporated under vacuum to give aresidue. The residue is taken up in 200 mL ethyl acetate, and washedsuccessively with 1N hydrochloric acid (1×50 mL) saturated sodiumbicarbonate 1×50 mL) and brine (1×50 mL). After drying with MgSO₄, thesolvent is evaporated under vacuum to give crude product.

The crude product is purified by column chromatography silica gel togive the title compound.

Example 96 Preparation of N-(1-butanesulfonyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid ##STR148##

The compound of Example 95 (1.55 g, 2.45 mmole) is dissolved in THF (50mL), 0.5 g of 10% palladium on carbon is added and the mixture isstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered from the reaction mixture, the solvent isremoved under vacuum and the residue is taken up in a solution ofsaturated sodium bicarbonate. This solution is then extracted with ethylacetate (1×150 mL) and the organic layer is decanted off. The remainingaqueous layer is layered with 100 mL of ethyl acetate and acidified with1N hydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 97 Preparation of N-(1-butanesulfonyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR149##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 96 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound. 0.1 mL ofthiocresol is additionally added to the anisole-HF cleavage mixture.

Example 98 Preparation of N-(2-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR150##

The compound of Example 94 (6.6 mmole) is dissolved in 12 mL of 4Nanhydrous hydrochloric acid/dioxane. The solution is stirred for severalhours at room temperature until all starting material is consumed. Thehydrochloric acid/dioxane solution is evaporated under vacuum. Theresidue is dissolved in acetonitrile and evaporated under vacuum. Thisis done three times to give a further residue.

The residue is suspended in 20 mL of acetonitrile, cooled to ice bathtemperature, then 2-naphthalenesulfonyl chloride (0.1.49 g, 6.6 mmole)and pyridine (1.57 g, 19.8 mmole) are added. The reaction is taken fromthe ice bath after 30 minutes and stirred at room temperature for 18hours. The acetonitrile is evaporated under vacuum to give residue whichis taken up in ethyl acetate (200 mL) and washed successively with 1Nhydrochloric acid (1×50 mL), saturated sodium bicarbonate (1×50 mL) andbrine (1×50 mL). The solution is dried with MgSO₄ and evaporated undervacuum will give crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 99 Preparation of N-(2-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid ##STR151##

The compound of Example 98 (3.58 mmole) is dissolved in THF (50 mL), 0.5g of 10% palladium on carbon is added and the mixture is stirred underhydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered off the reaction mixture, the solvent isremoved under vacuum to give a residue. The residue is taken up in asolution of saturated sodium bicarbonate, extracted with ethyl acetate(1×150 mL) and the organic layer is decanted off. The remaining aqueouslayer is layered with 100 mL of ethyl acetate and acidified with 1Nhydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 100 Preparation of N-(2-naphthalenesulfonyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR152##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 99 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 101 Preparation of N-benzylsulfonyl-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR153##

The compound of Example 94 (6.6 mmole) is dissolved in 12 mL of 4Nanhydrous hydrochloric acid/dioxane. The solution is stirred for severalhours at room temperature until all starting material is consumed. Thehydrochloric acid/dioxane solution is evaporated under vacuum. Theresidue is dissolved in acetonitrile and evaporated under vacuum. Thisis done three times to yield a further residue.

The residue is suspended in 20 mL of acetonitrile, cooled to ice bathtemperature, then alpha-toluenesulfonyl chloride (0.1.49 g, 6.6 mmole)and pyridine (1.57 g, 19.8 mmole) are added. The reaction is taken fromthe ice bath after 30 minutes and stirred at room temperature for 18hours. The acetonitrile is evaporated under vacuum to give residue whichis taken up in ethyl acetate (200 mL) and washed successively with 1Nhydrochloric acid (1×50 mL), saturated sodium bicarbonate (1×50 mL) andbrine (1×50 mL). The solution is dried with MgSO₄ and evaporated undervacuum to give crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 102 Preparation of N-benzylsulfonyl-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid ##STR154##

The compound of Example 101 (1.95 g, 3.58 mmole) is dissolved in THF (50mL), 0.5 g of 10% palladium on carbon is added and the mixture isstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered off the reaction mixture, the solvent isremoved under vacuum and the residue is taken up in a solution ofsaturated sodium bicarbonate. This solution is then extracted with ethylacetate (1×150 mL) and the organic layer is decanted off. The remainingaqueous layer is layered with 100 mL of ethyl acetate and acidified with1N hydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 103 Preparation of N-benzylsulfonyl-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR155##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 102 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 104 Preparation of N-(2-propylpentanoyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR156##

The compound of Example 94 (3.0 g, 4.66 mmole) is dissolved in 12 mL of4N anhydrous hydrochloric acid/dioxane. The solution is stirred forseveral hours at room temperature until all starting material isconsumed. The hydrochloric acid/dioxane solution is evaporated undervacuum to give a residue. This residue is dissolved in acetonitrile andevaporated under vacuum. This is done three times to yield a furtherresidue.

The residue is suspended in 25 mL of acetonitrile, cooled to ice bathtemperature, then 2-propylpentanoic acid (0.95 g, 6.6 mmole), BOP (2.92g, 6.6 mmole) and NMM (2.0 g, 19,8 mmole) are added. The reaction istaken from the ice bath after 30 minutes and stirred at room temperaturefor 18 hours. The acetonitrile is evaporated under vacuum to giveresidue which is taken up in ethyl acetate (200 mL) and washedsuccessively with 1N hydrochloric acid (1×50 mL), saturated sodiumbicarbonate (1×50 mL) and brine (1×50 mL). The solution is dried withMgSO₄ and evaporated under vacuum to give crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 105 Preparation of N-(2-propylpentanoyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid ##STR157##

The compound of Example 104 (1.75 g, 3.64 mmole) is dissolved in THF (50mL), 0.5 g of 10% palladium on carbon is added and the mixture isstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered off the reaction mixture, the solvent isremoved under vacuum and the residue is taken up in a solution ofsaturated sodium bicarbonate. This solution is then extracted with ethylacetate (1×150 mL) and the organic layer is decanted off. The remainingaqueous layer is layered with 100 mL of ethyl acetate and acidified with1N hydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 106 Preparation of N-(2-propylpentanoyl)-S-methyl-L-cysteinesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR158##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 105 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound. 0.1 mL ofthiocresol is additionally added to the anisole-HF cleavage mixture.

Example 107 Preparation of N-Boc-S-(t-butylacetate)-L-cysteine-L-2-azetidinecarboxylic acid-O-benzyl ester##STR159##

The compound of Example 51 (31.89 g, 95.06 mmole) and the compound of 80(95.06 mmole) are suspended in 140 mL of acetonitrile and 120 mL of DMFat 0° C., then BOP (42.0 g, 95.06 mmole) and NMM (28.84 g, 285.18 mmole)are added. The ice bath is removed after 30 minutes and the reaction isstirred for 18 hours at room temperature. The acetonitrile is evaporatedunder vacuum to give residue which is taken up in ethyl acetate (200 mL)and washed successively with 1N hydrochloric acid (1×50 mL), saturatedsodium bicarbonate (1×50 mL) and brine (1×50 mL). The solution is driedwith MgSO₄ and evaporated under vacuum to give crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 108 Preparation of N-Boc-S-(t butyl acetate)-L-cysteinesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR160##

The compound of Example 107 (55.07 mmole) is dissolved in 300 mL ofglacial acetic acid, sodium perborate tetrahydrate (42.36 g, 275.35mmole) is added and the mixture is heated to 55° C. After 2.5 hours atthis temperature, the reaction mixture is diluted with 1 liter of brine,the aqueous layer is extracted with ethyl acetate (4×250 mL) and thecombined organic extracts are dried with MgSO₄. The solution is filteredand evaporated under vacuum, then is repeatedly azeotroped with toluene(200 mL) under vacuum to remove acetic acid. The residue is dissolved inethyl acetate (200 mL), filtered and the filtrate is evaporated to givethe title compound.

Example 109 Preparation of N-benzylsulfonyl-S(t-butylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid-O-benzyl ester.##STR161##

A solution of the compound of Example 108 (9.28 mmole) in 105 mL ofsieve-dried ethyl acetate is prepared. To this, 26 mL of 5.7N anhydroushydrochloric acid/ethyl acetate (that is generated in situ from acetylchloride and methanol) is added. This mixture is stirred for severalhours at room temperature until all starting material is consumed. Themixture is evaporated under vacuum and the residue is dissolved inacetonitrile and then this solvent is removed under vacuum. This is donethree times to give a further residue.

The residue is suspended in 35 mL of acetonitrile, cooled to ice bathtemperature, then benzylsulfonyl chloride (2.12 g, 11.14 mmole) andpyridine (2.93 g, 37.12 mmole) are added. The reaction is removed fromthe ice bath after 30 minutes and allowed stirred at room temperaturefor 18 hours. The acetonitrile is evaporated under vacuum to give aresidue which is taken up in ethyl acetate (200 mL) and washedsuccessively with 1N hydrochloric acid (1×50 mL), saturated sodiumbicarbonate (1×50 mL) and brine (1×50 mL). The solution is dried withMgSO₄ and evaporated under vacuum to give crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 110 Preparation of N-benzylsulfonyl-S-(t-butylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid ##STR162##

The compound of Example 109 (4.81 mmole) is dissolved in THF (50 mL),0.5 g of 10% palladium on carbon is added and the mixture is stirredunder hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered off the reaction mixture, the solvent isremoved under vacuum and the residue is taken up in a solution ofsaturated sodium bicarbonate. This solution is then extracted with ethylacetate (1×150 mL) and the organic layer is decanted off. The remainingaqueous layer is layered with 100 mL of ethyl acetate and acidified with1N hydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 111 Preparation of N-benzylsulfonyl-S-(carboxymethyl)-L-cysteinesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR163##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 110 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 112 Preparation of N-(2-propylpentanoyl)-S-(t-butylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid-O-benzyl ester##STR164##

A solution of the compound of Example 108 (9.28 mmole) in 105 mL ofsieve-dried ethyl acetate is prepared. To this, 26 mL of 5.7N anhydroushydrochloric acid/ethyl acetate (that is generated in situ from acetylchloride and methanol) is added. This mixture is stirred for severalhours at room temperature until all starting material is consumed. Themixture is evaporated under vacuum and the residue is dissolved inacetonitrile and then this solvent is evaporated under vacuum. This isdone three times to give a further residue.

The residue is suspended in 35 mL of acetonitrile, cooled to ice bathtemperature, then 2-propylpentanoic acid (1.60 g, 11.4 mmole), BOP (4.10g, 9.28 mmole) and NMM (3.75 g, 37.1 mmole) are added. The reaction isremoved from the ice bath after 30 minutes and is allowed stir at roomtemperature for 18 hours. The reaction mixture is reduced in volumeunder vacuum to a residue.

The residue is taken up in 200 mL ethyl acetate, and is washedsuccessively with 1N hydrochloric acid (1×50 mL) saturated sodiumbicarbonate 1×50 mL) and brine (1×50 mL). After drying with MgSO₄, thesolvent is evaporated under vacuum to give crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 113 Preparation of N-(2-propylpentanoyl)-S-(t-butylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid ##STR165##

The compound of Example 112 (1.81 g, 3.2 mmole) is dissolved in THF (50mL), 0.5 g of 10% palladium on carbon is added and the mixture isstirred under hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered off the reaction mixture, the solvent isremoved under vacuum and the residue is taken up in a solution ofsaturated sodium bicarbonate. This solution is then extracted with ethylacetate (1×150 mL) and the organic layer is decanted off. The remainingaqueous layer is layered with 100 mL of ethyl acetate and acidified with1N hydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 114 Preparation ofN-(2-propylpentanoyl)-S-(carboxymethyl)-L-cysteinesulfone-L-2-azetidinecarboxylic acid-L-argininal ##STR166##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 113 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 115 Preparation of N-Boc-S-(methylacetate)-L-cysteine-L-2-azetidinecarboxylic acid-O-benzyl ester##STR167##

The compound of Example 61 (20.69 g, 70.5 mmole) and the compound ofExample 80 (70.5 mmole) are suspended in 140 mL of acetonitrile at 0°C., then BOP (30.96 g, 70.5 mmole) and NMM (21.2 g, 211 mmole) areadded. The ice bath is removed after 30 minutes and the reaction isstirred for 18 hours at room temperature. The reaction mixture isreduced in volume at 25° C. under vacuum to give a residue. The residueis taken up in 200 mL ethyl acetate, and is washed successively with 1Nhydrochloric acid (1×50 mL) saturated sodium bicarbonate 1×50 mL) andbrine (1×50 mL). After drying with MgSO₄, the solvent is evaporatedunder vacuum to give crude product.

This crude product is purified by column chromatography on silica gel togive the title compound.

Example 116 Preparation of N-Boc-S-(methyl acetate)-L-cysteinesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR168##

The compound of Example 115 (31.55 mmole) is dissolved in 165 mL ofglacial acetic acid, sodium perborate tetrahydrate (24.27 g, 157.75mmole) is added and the mixture is heated to 55° C. After 2.5 hours atthis temperature, the reaction solution is diluted with 700 mL of brine,the aqueous layer is extracted with ethyl acetate (3×250 mL) and thecombined organic phases are dried with MgSO₄. The solution is filteredand evaporated under vacuum, then is repeatedly azeotroped with toluene(200 mL) under vacuum to remove acetic acid. The residue is dissolved inethyl acetate (200 mL), filtered and the filtrate is evaporated undervacuum to give the title compound.

Example 117 Preparation of N-(2-naphthylenesulfonyl)-S-(methylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid-O-benzyl ester##STR169##

The compound of Example 116 (4.8 g, 9.37 mmole) is dissolved in 18 mL of4N anhydrous hydrochloric acid/dioxane. The solution is stirred forseveral hours at room temperature until all starting material isconsumed. The hydrochloric acid/dioxane solution is evaporated undervacuum to give a residue. The residue is dissolved in acetonitrile andevaporated under vacuum. This is done three times to give a furtherresidue.

The residue is suspended in 35 mL of acetonitrile, cooled to ice bathtemperature, then 2-naphthylenesulfonyl chloride (2.55 g, 11.2 mmole)and pyridine (2.2 g, 28.1 mmole) are added. The reaction is taken fromthe ice bath after 30 minutes and stirred at room temperature for 18hours. The residue is taken up in 200 mL ethyl acetate, and is washedsuccessively with 1N hydrochloric acid (1×50 mL) saturated sodiumbicarbonate 1×50 mL) and brine (1×50 mL). After drying with MgSO₄, thesolvent is evaporated under vacuum to give crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 118 Preparation of N-(2-naphthylenesulfonyl)-S-(methylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid ##STR170##

The compound of Example 117 (6.47 mmole) is dissolved in THF (50 mL),0.5 g of 10% palladium on carbon is added and the mixture is stirredunder hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered off the reaction mixture, the solvent isremoved under vacuum and the residue is taken up in a solution ofsaturated sodium bicarbonate. This solution is then extracted with ethylacetate (1×150 mL) and the organic layer is decanted off. The remainingaqueous layer is layered with 100 mL of ethyl acetate and acidified with1N hydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 119 Preparation of N-(2-naphthylenesulfonyl)-S-(methylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid-L-argininal##STR171##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 118 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

Example 120 Preparation of N-benzylsulfonyl-S-(methyl acetate-L-cysteinesulfone-L-2-azetidinecarboxylic acid-O-benzyl ester ##STR172##

The compound of Example 116 (10.9 mmole) is dissolved in 18 mL of 4Nanhydrous hydrochloric acid/dioxane. The solution is stirred for severalhours at room temperature until all starting material is consumed. Thehydrochloric acid/dioxane solution is evaporated under vacuum to give aresidue. The residue is dissolved in acetonitrile and evaporated undervacuum. This is done three times to give a further residue.

The residue is suspended in 25 mL of acetonitrile, cooled to ice bathtemperature, then benzylsulfonyl chloride (2.5 g, 13.1 mmole) andpyridine (2.6 g, 32.8 mmole) are added. The reaction is taken from theice bath after 30 minutes and stirred at room temperature for 18 hours.The reaction mixture is reduced in volume under vacuum to a residue. Theresidue is taken up in 200 mL ethyl acetate, and is washed successivelywith 1N hydrochloric acid (1×50 mL) saturated sodium bicarbonate 1×50mL) and brine (1×50 mL). After drying with MgSO₄, the solvent isevaporated under vacuum to give crude product.

The crude product is purified by column chromatography on silica gel togive the title compound.

Example 121 Preparation of N-benzylsulfonyl-S-(methylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid ##STR173##

The compound of Example 120 (5.12 mmole) is dissolved in THF (50 mL),0.5 g of 10% palladium on carbon is added and the mixture is stirredunder hydrogen gas at atmospheric pressure for 18 hours.

After the catalyst is filtered off the reaction mixture, the solvent isremoved under vacuum and the residue is taken up in a solution ofsaturated sodium bicarbonate. This solution is then extracted with ethylacetate (1×150 mL) and the organic layer is decanted off. The remainingaqueous layer is layered with 100 mL of ethyl acetate and acidified with1N hydrochloric acid to pH 2 (pH papers). After the phases separate, theorganic layer is saved and the aqueous layer is then further extractedwith ethyl acetate (3×100 mL).

The organic extracts are combined and washed with brine, dried withMgSO₄, filtered and evaporated under vacuum to give the title compound.

Example 122 Preparation of N-benzylsulfonyl-S-(methylacetate)-L-cysteine sulfone-L-2-azetidinecarboxylic acid-L-argininal##STR174##

The title compound is prepared in the same manner as described inExample 8.

The compound of Example 121 is attached to the resin of Example 7. Thetitle compound is cleaved from the resin as a protected semicarbazone.The semicarbazone is hydrolyzed to give the title compound.

By following the procedures of the Example 1 to 122, the followingcompounds are made: ##STR175##

Example A Kinetic Analysis of Selected Compounds in an in vitro ThrombinInhibition Assay

1. Determination of IC₅₀.

The ability of the compounds of the present invention to act asinhibitors of thrombin and plasmin catalytic activity was assessed bydetermining the concentration which inhibited enzyme activity by 50%,(IC₅₀), using the purified human enzymes.

The buffer used for all assays was HBSA (10 mM HEPES, pH 7.5, 150 mMsodium chloride, 0.1% bovine serum albumin).

The assay for IC₅₀ determinations was conducted by combining inappropriate wells of a Corning microtiter plate, 50 microliters of HBSA,50 microliters of the nest compound at a specified concentration(covering a broad concentration range) diluted in HBSA (or HBSA alonefor V_(o) (uninhibited velocity) measurement), and 50 microliters of theenzyme diluted in HBSA. Following a 30 minute incubation at ambienttemperature, 50 microliters of the substrate at the concentrationsspecified below, was added to the wells yielding a final total volume of200 microliters. The initial velocity of chromogenic substratehydrolysis was measured by the change in absorbance at 405 nm using aThermo Max® Kinetic Microplate Reader over a 5 minute period in whichless than 5% of the added substrate was utilized. The concentration ofadded inhibitor which caused a 50% decrease in the initial rate ofhydrolysis was defined as the IC₅₀ value.

Thrombin Assay

Thrombin catalytic activity was determined using the chromogenicsubstrate Pefachrome t-PA (CH₃ SO₂-D-hexahydrotyrosine-glycyl-L-arginine-p-nitroaniline, obtained fromPentapharm Ltd.). The substrate was made up in deionized water followedby dilution in HBSA prior to the assay in which the final concentrationwas 300 μM (about 10-times Km). Purified human alpha-thrombin wasobtained from Enzyme Research Laboratories, Inc. The enzyme was dilutedinto HBSA prior to the assay in which the final concentration was 0.25nM.

Factor Xa Assay

Factor Xa catalytic activity was determined using the substrate, S2765(N-alpha-benzyloxycarbonyl-D-argininyl-L-glycyl-L-arginine-p-nitroanilidedihydrochloride) which was obtained from Kabi Diagnostica. The substratewas made up in deionized water followed by dilution in HBSA prior to theassay in which the final concentration was 250 μM (about 5-times Km).Human factor Xa was prepared from purified human factor X obtained fromEnzyme Research Laboratories according to the method described by Bock,P. E. et al., Archives of Biochem. Biophys. 273:375 (1989). The enzymewas diluted into HBSA prior to the assay in which the finalconcentration was 0.5 nM.

Plasmin Assay

Plasmin catalytic activity was determined using the chromogenicsubstrate, S-2251 [D-valyl-L-leucyl-L-lysine-p-nitroanilidedihydrochloride], which was obtained from Kabi Diagnostica. Thesubstrate was made up in deionized water followed by dilution in HBSAprior to the assay in which the final concentration was 300 μM (about5-times Km). Purified human plasmin was obtained from Enzyme ResearchLaboratories, Inc. The enzyme was diluted into HBSA prior to assay inwhich the final concentration was 1.0 nM.

Table I below gives the IC₅₀ values for thrombin and plasmin forselected test compounds of the present invention.

                                      TABLE 1                                     __________________________________________________________________________    IC.sub.50 of Preferred Compounds.                                                                      From IC50, micromolar                                Compound Tested          Example                                                                            Thrombin                                                                            Plasmin                                   __________________________________________________________________________    BuSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                              11   0.0016                                                                              0.199                                     ChxCH.sub.2 SO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                    18   0.00066                                                                             0.030                                     2-CMPhSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                          --   0.00081                                                                             0.20                                      1-NpSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                            21   0.00078                                                                             >0.025                                    2-NpSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                            24   0.00058                                                                             0.035                                     BzlSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                             27   0.0010                                                                              0.086                                     d-Camphor--SO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                     --   0.0026                                                                              0.030                                     ChxNH--SO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                         --   0.146 2.6                                       2-PrPen--Met[S(O.sub.2)]--Pro--Arg-al                                                                  33   0.0055                                                                              0.50                                      Bzl--SO.sub.2 --Cys[S--CH.sub.2 CO.sub.2 H]--Pro--Arg-al                                               --   0.013 0.49                                      BuSO.sub.2 --Cys[S(O.sub.2)--CH.sub.3 ]--Pro--Arg-al                                                   40   0.0018                                                                              >0.25                                     2-PrPen--Cys[S(O.sub.2)--CH.sub.3 ]--Pro--Arg-al                                                       43   0.0051                                                                              2.8                                       2-NpSO.sub.2 --Cys[S(O.sub.2)--CH.sub.3 ]--Pro--Arg-al                                                 46   0.0014                                                                              >0.25                                     1-NpSO.sub.2 --Cys[S(O.sub.2)--CH.sub.3 ]--Pro--Arg-al                                                 49   0.0031                                                                              0.25                                      Bu--SO.sub.2 --Cys[S(O.sub.2)--CH.sub.2 CO.sub.2 H]--Pro--Arg-al                                       --   0.0030                                                                              0.11                                      BzlSO.sub.2 --Cys[S(O.sub.2)--CH.sub.2 CO.sub.2 H]--Pro--Arg-al                                        56   0.013 0.49                                      2-PrPen--Cys[S(O.sub.2)--CH.sub.2 CO.sub.2 H]--Pro--Arg-al                                             59   0.0016                                                                              0.22                                      2-NpSO.sub.2 --Cys[S(O.sub.2)--CH.sub.2 CO.sub.2 CH.sub.3 ]--Pro--Arg-al                               69   0.00056                                                                             0.061                                     __________________________________________________________________________

2. Determination of Inhibition constant, Ki.

The ability of the compounds of the present invention to act asinhibitors of thrombin catalytic activity was assessed by determiningtheir inhibition constant, Ki.

Enzyme activity was determined using the chromogenic substratePefachrome t-PA (CH₃ SO₂-D-hexahydrotyrosine-glycyl-L-arginine-p-nitroaniline), obtained fromPentapharm Ltd. The substrate was reconstituted in deionized water priorto use. Purified human alpha-thrombin (3000U/mg specific activity) wasobtained from Enzyme Research Laboratories, Inc. The buffer used for allassays was HBSA (10 mM HEPES, pH 7.5, 150 mM sodium chloride, 0.1%bovine serum albumin).

The assay for Ki determinations was conducted by combining inappropriate wells of a Corning microtiter plate, 50 microliters of HBSA,50 microliters of the test compound at a specified concentration dilutedin HBSA (or HBSA alone for V_(o) (uninhibited velocity) measurement),and 50 microliters of the chromogenic substrate (250 micromolar, 5×Km)At time zero, 50 microliters of alpha-thrombin diluted in HBSA, wasadded to the wells yielding a final concentration of 0.5 nM in a totalvolume of 200 microliters. Velocities of chromogenic substratehydrolysis which occurred over 40 minutes was measured by the change inabsorbance at 405nm using a Thermo Max® Kinetic Microplate Reader.

Ki values were determined for test compounds using the relationshipsdeveloped by Williams and Morrison, Methods in Enzymology, 63:437 (1979)using steady state velocities (Vs) measured over 40 minutes. The extentof substrate hydrolysis was less than 5% over the course of this assay.

Table 2 below gives the Ki values for selected test compounds. The datashows their utility as patent in vitro inhibitors of humanalpha-thrombin.

                  TABLE 2                                                         ______________________________________                                        Inhibitor Constants (Ki) of Preferred Compounds.                              Compound               Ki (nM)                                                ______________________________________                                        2-PrPen--Met[S(O.sub.2)]--Pro--Arg-al.sup.1                                                          4.0                                                    2-NpSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al.sup.2                                                    0.135                                                  BzlSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al.sup.3                                                     1.06                                                   ______________________________________                                         .sup.1 This is the compound of Example 33.                                    .sup.2 This is the compound of Example 24.                                    .sup.3 This is the compound of Example 27.                               

Example B Ex vivo Anticoagulant Effects of BzlSO₂ -Met[S(O₂)]-Pro-Arg-alin Rat and Human Plasma

The ex vivo anticoagulant effects of BzlSO₂ -Met[S(O₂)]-Pro-Arg-al wasdetermined by measuring the prolongation of the activated partialthromboplastin time (APTT) over a broad concentration range of the addedinhibitor, using pooled normal human and rat plasma. Fresh frozencitrated pooled normal human plasma was obtained from George KingBiomedical, Overland Park, Kans. Pooled normal rat plasma was preparedfrom citrated whole blood collected from anesthetized rats usingstandard procedures. The plasma was flash frozen and stored at -80° C.until use. Measurements APTT was made using the Coag-A-Mate RA4automated coagulometer (General Diagnostics, Organon Technica, OklahomaCity, Okla.) using the Automated APTT reagent (Organon Technica, Durham,N.C.) as the initiator of clotting according to the manufacturer'sinstructions.

The assay was conducted by making a series of dilutions of the testcompounds in rapidly thawed plasma followed by adding 200 microliters tothe wells of the assay carousel.

FIG. 2 shows the anticoagulant effect of BzlSO₂ -Met[S(O₂)]-Pro-Arg-alwas measured in citrated rat () and human (∘) plasma using thisactivated partial thromboplastin time (APTT) assay. The elevation inAPTT due to increasing concentration of compound is presented relativeto the control clotting time for rat (19.75 sec) and human (28.3 sec)plasma which is set to a value of 1.0. It is clear from these data thatBzlSO₂ -Met[S(O₂)]-Pro-Arg-al prolonged the APTT in a dose dependentmanner in both rat and human plasma indicating a similar anticoagulanteffect in both species of mammals.

Example C Evaluation of the Antithrombotic Potential of BzlSO₂-Met[S(O₂)]-Pro-Arg-al in an Experimental Rat Model of Thrombosis

The demonstrated anticoagulant effects of BzlSO₂ -Met[S(O₂)]-Pro-Arg-alin both rat and human citrated plasma indicated that this compound mayhave potent antithrombotic effects in an experimental model ofthrombosis performed in rats. To investigate this, the antithrombotic(prevention of thrombus formation) properties of this compound wasevaluated using the following established experimental model of acutevascular thrombosis.

Rat model of FeCl₃ -induced platelet-dependent arterial thrombosis

This is a well characterized model of platelet dependent, arterialthrombosis which has been used in the evaluation potentialantithrombotic compounds such as direct thrombin inhibitors. Kurz, K.D., Main, B. W., and Sandusky, G. E., Thromb. Res., 60:269-280 (1990).In this model a platelet-rich, occlusive thrombus is formed in a segmentof the rat carotid artery treated locally with a fresh solution of FeCl₃absorbed to a piece of filter paper. The FeCl₃ is thought to diffuseinto the treated segment of artery and causes de-endothelialization ofthe affected vessel surface. This results in the exposure of blood tosubendothelial structures which in turn causes platelet adherence,thrombin formation and platelet aggregation resulting in occlusivethrombus formation. The effect of a test compound on the incidence ofocclusive thrombus formation following the application of the FeCl₃ ismonitored by ultrasonic flowtometry and is used as the primary endpoint. The use of flowtometry to measure carotid artery blood flow, is amodification of the original procedure in which thermal detection ofclot formation was employed. Kurz, K. D., Main, B. W., and Sandusky, G.E., Thromb. Res., 60:269-280 (1990).

Male Harlan Sprague Dawley rats (420-450 g) were acclimated at least 72hours prior to use and fasted for 12 hours prior to surgery with freeaccess to water. The animals were prepared, anesthetized with Nembutalfollowed by the insertion of catheters for blood pressure monitoring,drug and anesthesia delivery. The left carotid artery was isolated bymaking a midline cervical incision followed by blunt dissection andspreading techniques to separate a 2 cm segment of the vessel from thecarotid sheath. A silk suture was inserted under the proximal and distalends of the isolated vessel to provide clearance for the placement of aultrasonic flow probe (Transonic) around the proximal end of the vessel.The probe was then secured with a stationary arm.

Following surgery the animals were randomized in either a control(saline) or treatment group with test compound (BzlSO₂-Met[S(O₂)]-Pro-Arg-al) with at least 6 animals per group per dose. Thetest compound was administered as a single intravenous bolus at thedoses outlined in Table 3 after placement of the flow probe and 5minutes prior to the application of the FeCl₃ thrombogenic stimulus(t=0). At t=0, a 3 mm diameter piece of filter paper (Whatman #3) soakedwith 10 microliters of a 35% solution of fresh FeCl₃ (made up in water)was applied the segment of isolated carotid artery distal to the flowprobe. Blood pressure, blood flow, heart rate, and respiration weremonitored for 60 minutes.

The incidence of occlusion (defined as the attainment of zero bloodflow) was recorded as the primary end point.

The efficacy of BzlSO₂ -Met[S(O₂)]-Pro-Arg-al as an antithrombotic agentin preventing thrombus formation in this in vivo model was demonstratedby the reduction in the incidence of thrombotic occlusion as shown inTable 3 below.

                  TABLE 3                                                         ______________________________________                                        Results of BzlSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                         (Test Cmpd) in the FeCl.sub.3 Model of Thrombosis in Rats.                    Treatment Dose                Incidence of                                    Group     (mg/kg)       n     Occlusion                                       ______________________________________                                        Saline    --            6     6/6                                             Test Cmpd 0.5           6     6/6                                             Test Cmpd  0.75         6     2/6                                             Test Cmpd 1.0           6      1/6*                                           Test Cmpd 3.0           6      0/6*                                           ______________________________________                                         *-p ≦ 0.05 from saline control by Fishers test                    

The therapeutically effective amount (dose) which prevents 50% ofthrombotic occlusions in this model (ED₅₀) can be determined from theabove data by plotting the incidence of occlusion Versus the doseadministered. This allows a direct comparison of the antithromboticefficacy of BzlSO₂ -Met[S(O₂ ]-Pro-Arg-al with other antithromboticagents which have also been evaluated in this model as described above.Table 4 lists the ED₅₀ values for several well known anticoagulantagents in this model compared to this compound.

                  TABLE 4                                                         ______________________________________                                        Efficacy of BzlSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al compared to            other antithrombotic agents based on ED.sub.50 for thrombus                   prevention in the FeCl.sub.3 model of arterial thrombosis.                                        Ki      ED.sub.50.sup.b                                   Compound            (nM)    (mg/kg)                                           ______________________________________                                        Standard Heparin            300     U/kg                                      Argatroban          19.0.sup.c                                                                            3.8     mg/kg                                     Hirulog ™        2.56.sup.d                                                                            3.0     mg/kg                                     BzlSO.sub.2 --Met[S(O.sub.2)]--Pro--Arg-al                                                        1.06.sup.a                                                                            0.7     mg/kg                                     ______________________________________                                         .sup.a Ki determined using human alphathrombin as described above and in      items c and d                                                                 .sup.b ED.sub.50 is defined as the dose that prevents the incidence of        complete thrombotic occlusion in 50% of animals tested                        .sup.c Kikumoto, R. et. al. Biochemistry 23: 85-90 (1984)                     .sup.d Witting, J. I. et. al. Biochem. J. 283: 737-743 (1992)            

The data presented in Table 4 clearly demonstrates the effectiveness ofBzlSO₂ -Met[S(O₂)]-Pro-Arg-al in preventing occlusive thrombus formationin this experimental model. The relevance of this data to preventinghuman thrombosis can be inferred from the comparison to the otheranticoagulant agents listed in this table which have been evaluated inan identical manner in this experimental model and have demonstratedantithrombotic efficacy in preventing thrombus formation clinically asdescribed in the following literature citations: Heparin-Hirsh, J. N.Engl., J. Med 324:1565-1574 (1992) and Cairns, J. A. et. al., Chest,102:456S-481S (1992); Argatroban-Gold, H. K. et.al., J. Am. Coll.Cardiol., 21:1039-1047 (1993); and Hirulog™-Sharma, G.V.R.K. et.al., Am.J. Cardiol., 72:1357-1360 (1993) and Lidon, R. M. et.al., Circulation,88:1495-1501 (1993).

The in vivo comparison of BzlSO₂ -Met[S(O₂)]-Pro-Arg-al with theclinically effective antithrombotic agents Heparin, Argatroban, andHirulog™ in the same rodent model of experimental thrombosis coupledwith the demonstrated anticoagulant effects of BzlSO₂-Met[S(O₂)]-Pro-Arg-al in both rat and human plasma described above inExample B clearly suggests that this compound will be an effectiveantithrombotic agent in humans.

We claim:
 1. A compound of the formula: ##STR176## (a) X is selectedfrom the group consisting of --C(O)--, --S(O₂)--, --O--S(O₂)--,--NH--S(O₂)-- and --N(R')--S(O₂)--, wherein R' is alkyl of 1 to about 4carbon atoms, aryl of about 6 to about 14 carbon atoms, or aralkyl ofabout 6 to about 15 carbon atoms;(b) R₁ is selected from the groupconsisting of:(1) alkyl of about 3 to about 10 carbon atoms, (2) alkylof 1 to about 3 carbon atoms substituted with cyclic alkyl of about 5 toabout 8 carbon atoms, (3) alkenyl of about 3 to about 6 carbon atoms,(4) alkenyl of about 3 to about 6 carbon atoms which is substituted withcyclic alkyl of about 5 to about 8 carbon atoms, (5) aryl of about 6 toabout 14 carbon atoms, (6) aryl of about 6 to about 14 carbon atomswhich is substituted with Y₁, (7) aryl of about 6 to about 14 carbonatoms which is substituted with Y₁ and Y₂, (8) aralkyl of about 6 toabout 15 carbon atoms, (9) aralkyl of about 6 to about 15 carbon atomswhich is substituted in the aryl ring with Y₁, (10) aralkyl of about 6to about 15 carbon atoms which is substituted in the aryl ring with Y₁and Y₂, (11) aralkenyl of about 8 to about 15 carbon atoms, (12)aralkenyl of about 8 to about 15 carbon atoms which is substituted inthe aryl ring with Y₁, (13) aralkenyl of about 8 to about 15 carbonatoms which is substituted in the aryl ring with Y₁ and Y₂, (14)perfluoroalkyl of 1 to about 12 carbon atoms, (15) perfluoroaryl ofabout 6 to about 14 carbon atoms, (16) trimethylsilylalkyl of about 4 toabout 8 carbon atoms, ##STR177## wherein Y₁ and Y₂ are independentlyselected from the group consisting of bromo, chloro, fluoro, --Z₁, --OH,--OZ₁, --NH₂, --NHZ₁, --NZ₁ Z₂, --NH--C(O)--Z₁, --N(Z₁)--C(O)--Z₂,--NH--C(O)--OZ₁, --N(Z₁)--C(O)--OZ₂, --NH--C(O)--NH₂, --NH--C(O)--NHZ₁,--NH--C(O)--NZ₁ Z₂, --N (Z₁)--C(O)--NHZ₂, --N(Z₁)--C(O)--NZ₂ Z₃,--C(O)--OH, --C(O)--OZ₁, --C(O)--NHZ₁, --C(O)--NZ₁ Z₂, --SH, --SZ₁,--S(O)--Z₁, --S(O₂)--Z₁, --S(O₂)--OH, --S(O₂)--OZ₁, --S(O₂ --NH₂,--S(O₂)--NHZ₁, --S(O₂)--NZ₁ Z₂ and ##STR178## wherein Z₁, Z₂ and Z₃ areindependently selected from the group consisting of trifluoromethyl,pentafluoroethyl, alkyl of 1 to about 12 carbon atoms, aryl of about 6to about 14 carbon atoms, and aralkyl of about 6 to about 15 carbonatoms,R' is alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about14 carbon atoms, or aralkyl of about 6 to about 15 carbon atoms, (c) R₂is selected from the group consisting of

    --CH.sub.2 --S--CH.sub.3,

    --CH.sub.2 --S(O)--CH.sub.3,

    --CH.sub.2 --S(O.sub.2)--CH.sub.3,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OH,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OR',

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NHR',

    --CH.sub.2 --S--(CH.sub.2).sub.m --C(O)--NR'R",

    --CH.sub.2 --S(O)--(CH.sub.2).sub.m --C(O)--NR'R",

    --CH.sub.2 --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NR'R", ##STR179##

    --S--CH.sub.3,

    --S(O)--CH.sub.3,

    --S(O.sub.2)--CH.sub.3,

    --S--(CH.sub.2).sub.m --C(O)--OH,

    --S(O)--(CH.sub.2).sub.m --C(O)--OH,

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OH,

    --S--(CH.sub.2).sub.m --C(O)--OR',

    --S(O)--(CH.sub.2).sub.m --C(O)--OR',

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--OR',

    --S--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S(O)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NH.sub.2,

    --S--(CH.sub.2).sub.m --C(O)--NHR',

    --S(O)--(CH.sub.2).sub.m --C(O)--NHR',

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NHR',

    --S--(CH.sub.2).sub.m --C(O)--NR'R",

    --S(O)--(CH.sub.2).sub.m --C(O)--NR'R",

    --S(O.sub.2)--(CH.sub.2).sub.m --C(O)--NR'R", ##STR180## wherein is 1, 2, 3, 4, 5 or 6; and (d) n is 3; or pharmaceutically acceptable salts thereof.


2. A compound of claim 1 wherein R₂ is --S(O)₂ CH₂ C(O)OH, --S(O)₂ CH₂C(O)OCH₃, --S(O)₂ CH₂ C(O)OCH₂ CH₃ or --S(O)₂ CH₂ C(O)OCH₂ CH₂ CH₃.
 3. Acompound of claim 2 wherein X is --S(O₂)--, --NH--S(O₂)-- or --C(O)--.4. A compound of claim 3 wherein X is --S(O₂)--.
 5. A compound of claim4 wherein R₁ is alkyl of about 3 to about 10 carbon atoms, alkyl of 1 toabout 3 carbon atoms substituted with cyclic alkyl of about 5 to about 8carbon atoms, aryl of about 6 to about 14 carbon atoms which isoptionally mono-substituted with Y₁ or optionally di-substituted with Y₁and Y₂ or aralkyl of about 6 to about 15 carbon atoms which isoptionally monosubstituted on the aryl ring with Y₁ or optionallydi-substituted on the aryl ring with Y₁ and Y₂.
 6. A compound of claim 3wherein X is --C(O)--.
 7. A compound of claim 6 wherein R₁ is alkyl ofabout 3 to about 10 carbon atoms, alkyl of 1 to about 3 carbon atomssubstituted with cyclic alkyl of about 5 to about 8 carbon atoms oraralkyl of about 6 to about 15 carbon atoms which is optionallymono-substituted on the aryl ring with Y₁ or optionally di-substitutedon the aryl ring with Y₁ and Y₂.
 8. A compound according to claim 7wherein R₂ is --S(O)₂ CH₂ C(O)OCH₃.
 9. A compound according to claim 8wherein R₁ is 4-heptyl.
 10. A compound according to claim 1 wherein R₂is --CH₂ S(O)₂ CH₃.
 11. A compound according to claim 10 wherein X is--S(O₂)-- or --C(O)--.
 12. A compound according to claim 11 wherein X is--S(O₂)--.
 13. A compound according to claim 12 wherein R₁ is alkyl ofabout 3 to about 10 carbon atoms, alkyl of 1 to about 3 carbon atomssubstituted with cyclic alkyl of about 5 to about 8 carbon atoms, arylof about 6 to about 14 carbon atoms which is optionally mono-substitutedwith Y₁ or optionally di-substituted with Y₁ and Y₂, or aralkyl of about6 to about 15 carbon atoms which is optionally mono-substituted on thearyl ring with Y₁ or optionally di-substituted on the aryl ring with Y₁and Y₂.
 14. A compound according to claim 11 wherein X is --C(O)--. 15.A compound according to claim 15 wherein R₁ is alkyl of about 3 to about10 carbon atoms, alkyl of 1 to about 3 carbon atoms substituted withcyclic alkyl of about 5 to about 8 carbon atoms, aryl of about 6 toabout 14 carbon atoms which is optionally mono-substituted with Y₁ oroptionally di-substituted with Y₁ and Y₂, or aralkyl of about 6 to about15 carbon atoms which is optionally mono-substituted on the aryl ringwith Y₁ or optionally di-substituted on the aryl ring with Y₁ and Y₂.16. A compound according to claim 1 wherein R₂ is --S(O)₂ CH₃.
 17. Acompound according to claim 16 wherein X is --S(O₂)-- or --C(O)--.
 18. Acompound according to claim 17 wherein X is --S(O₂)--.
 19. A compoundaccording to claim 18 wherein R₁ is alkyl of about 3 to about 10 carbonatoms, alkyl of 1 to about 3 carbon atoms substituted with cyclic alkylof about 5 to about 8 carbon atoms, aryl of about 6 to about 14 carbonatoms which is optionally mono-substituted with Y₁ or optionallydi-substituted with Y₁ and Y₂, and aralkyl of about 6 to about 15 carbonatoms which is optionally mono-substituted on the aryl ring with Y₁ oroptionally di-substituted on the aryl ring with Y₁ and Y₂.
 20. Apharmaceutical composition for the prevention of thrombosis in a mammalsuspected of having a condition characterized by abnormal thrombosis,comprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of the compound of claim 1, 2, 10, or
 16. 21. A methodfor the prevention of thrombosis in a mammal suspected of having acondition characterized by abnormal thrombosis, comprising administeringto said mammal a therapeutically effective amount of the compound of 1,2, 10 or
 16. 22. A method for the prevention of thrombosis in a mammalsuspected of having a condition characterized by abnormal thrombosis,comprising administering to said mammal a therapeutically effectiveamount of the pharmaceutical composition of claim 20.